Rnai target gene that is highly lethal to aphids and use thereof

ABSTRACT

Provided are a RNAi target gene that is lethal to aphids and the use thereof. Specifically, provided are six gene fragments resulting in the death of aphid nymphs and/or death thereof in the adult stage based on RNA interference technology. The death of the aphids can be caused by spraying a dsRNA-containing composition onto plants to feed aphids or directly spraying same onto the skin of the aphids.

TECHNICAL FIELD

The present invention belongs to the fields of biotechnology andagricultural applications. Specifically, the present invention relatesto RNAi target genes that are highly effective in killing aphids anduses thereof.

BACKGROUND

Aphid is an important worldwide pest. It belongs to Hemiptera,Aphidoidea. At present, more than 4,700 kinds of aphids are known. Theyare small in size and fast in reproduction. They are importantagricultural and horticultural pests. For the prevention and control ofaphids, currently it is still dominated by chemical agents. However, dueto its fast reproduction speed and strong concealment, its controleffect is poor, and a large amount of pesticides are required to inhibitits reproduction, which inevitably leads to resistance of aphids.

RNAi is widely used as a tool for gene function research, especially inanimals and plants with imperfect genetic manipulation tools. However,currently in insects, after dsRNA enters the insect body throughfeeding, it must enter the cell to activate the RNAi mechanism. Insectintestinal wall cells can prevent most dsRNA from entering othertissues, which is a key factor affecting the efficiency of RNAi, and isalso the biggest obstacle in the application of dsRNA oral deliverymethods.

Because different kinds of insects have different dsRNA uptakemechanisms, leading to differences in their response to dsRNA and targetgene silencing efficiency. Therefore, the lethal effects of differentkinds of insects are quite different.

Therefore, there is an urgent need in the art to develop an RNAi targetgene that is highly effective in killing aphids.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an RNAi target genethat is highly effective in killing aphids.

In a first aspect of the present invention, it provides a dsRNAconstruct, the dsRNA construct is double-stranded, and its positive ornegative strand contains a structure as shown in Formula I:

Seq_(forward)-X-Seq_(reverse)  Formula I

wherein

Seq_(forward) is a nucleotide sequence of insect nymph and/or adultstage regulation-related gene or fragment;

Seq_(reverse) is a nucleotide sequence that is basically complementaryto Seq_(forward);

X is an intervening sequence between the Seq_(forward) and theSeq_(reverse), and the intervening sequence is not complementary to theSeq_(forward) and the Seq_(reverse),

wherein the insect nymph and/or adult stage regulation-related gene isselected from the group consisting of DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, DS45 gene and a combination thereof.

In another preferred embodiment, the length of the dsRNA is at least21nt.

In another preferred embodiment, for the DS7 gene, the length of thedsRNA is 21nt-1350nt, preferably 506nt-1093nt.

In another preferred embodiment, for the DS9 gene, the length of thedsRNA is 21nt-909nt, preferably 54nt-631nt.

In another preferred embodiment, for the DS15 gene, the length of thedsRNA is 21nt-2148nt, preferably 516nt-1029nt.

In another preferred embodiment, for the DS25 gene, the length of thedsRNA is 21nt-1233nt, preferably 58nt-674nt.

In another preferred embodiment, for the DS27 gene, the length of thedsRNA is 21nt-1152nt, preferably, 219nt-748nt.

In another preferred embodiment, for the DS45 gene, the length of thedsRNA is 21nt-909nt, preferably 42nt-637nt.

In another preferred embodiment, the homology with the dsRNA is at least80%, preferably, 85%-100%.

In another preferred embodiment, the length of the Seq_(forward) and theSeq_(reverse) is at least 50 bp.

In another preferred embodiment, the dsRNA construct can form a dsRNA asshown in Formula II,

wherein

Seq′_(forward) is a RNA sequence or sequence fragment corresponding tothe Seq_(forward) sequence;

Seq′_(reverse) is a sequence that is basically complementary to theSeq′_(forward);

X′ is none; or is an intervening sequence located between Seq′_(forward)and Seq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse),

∥ represents the hydrogen bond formed between Seq_(forward) andSeq_(reverse).

In another preferred embodiment, the dsRNA is dsRNA without loop.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 9-10.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 11-12.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 13-14.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 15-16.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 17-18.

In another preferred embodiment, the dsRNA is amplified from thesequence as shown in SEQ ID NO. 19-20.

In a second aspect of the present invention, it provides a dsRNA asshown in Formula II,

wherein

Seq′_(forward) is a RNA sequence or sequence fragment corresponding to anucleotide sequence of an insect nymph and/or adult stageregulation-related gene or fragment;

Seq′_(reverse) is a sequence that is basically complementary to theSeq′_(forward); X′ is none; or is an intervening sequence betweenSeq′_(forward) and Seq′_(reverse); and the intervening sequence is notcomplementary to Seq′_(forward) and Seq′_(reverse);

wherein, the insect nymph and/or adult stage regulation-related gene isselected from the group consisting of DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, DS45 gene, and a combination thereof;

∥ represents the hydrogen bond formed between Seq_(forward) andSeq_(reverse).

In another preferred embodiment, the length of the Seq_(forward) andSeq_(reverse) is at least 50 bp.

In another preferred embodiment, the length of the intervening sequenceX′ is 0-300 bp.

In another preferred embodiment, the nymph and/or adult stageregulation-related gene is derived from the Aphis.

In another preferred embodiment, the sequence of the DS7 gene is shownin SEQ ID NO. 1 or 24.

In another preferred embodiment, the sequence of the DS9 gene is shownin SEQ ID NO. 2 or 25.

In another preferred embodiment, the sequence of the DS15 gene is shownin SEQ ID NO. 3 or 26.

In another preferred embodiment, the sequence of the DS25 gene is shownin SEQ ID NO. 4 or 27.

In another preferred embodiment, the sequence of the DS27 gene is shownin SEQ ID NO. 5 or 28.

In another preferred embodiment, the sequence of the DS45 gene is shownin SEQ ID NO. 6 or 29.

In another preferred embodiment, the insect is a phytophagous insect,preferably a homoptera insect, most preferably Aphis.

In another preferred embodiment, the insect is selected from the groupconsisting of green peach aphid, soybean aphid, and a combinationthereof.

In a third aspect of the present invention, it provides an expressionvector containing the dsRNA construct according to the first aspect ofthe present invention.

In a fourth aspect of the present invention, it provides a host cellthat contains the expression vector according to the third aspect of thepresent invention or the DNA sequence corresponding to the dsRNAconstruct according to the first aspect of the present invention isintegrated into the chromosome.

In another preferred embodiment, the host cell is a plant cell,preferably a green leaf plant cell.

In another preferred embodiment, the plant includes a cruciferous plant(such as a vegetable or soybean).

In a fifth aspect of the present invention, it provides a compositioncomprising the dsRNA construct according to the first aspect of thepresent invention and/or the dsRNA according to the second aspect of thepresent invention, and an acceptable carrier for insect feeding.

In another preferred embodiment, the acceptable carrier for insectfeeding includes water.

In another preferred embodiment, the composition is a composition usedto induce or cause the death of aphis nymphs and/or adult stage.

In another preferred embodiment, the dsRNA has the following sequence:

dsRNA1: having a sequence corresponding to SEQ ID NO. 1 or 24;

dsRNA2: having a sequence corresponding to SEQ ID NO. 2 or 25;

dsRNA3: having a sequence corresponding to SEQ ID NO. 3 or 26;

dsRNA4: having a sequence corresponding to SEQ ID NO. 4 or 27;

dsRNA5: having a sequence corresponding to SEQ ID NO. 5 or 28;

dsRNA6: having a sequence corresponding to SEQ ID NO. 6 or 29.

In another preferred embodiment, the DS7 gene, DS9 gene, DS15 gene, DS25gene, DS27 gene, and/or DS45 gene is from an insect, preferably from aHomoptera insect, and most preferably from Aphis.

In another preferred embodiment, the content of dsRNA1 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In another preferred embodiment, the content of dsRNA2 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In another preferred embodiment, the content of dsRNA3 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In another preferred embodiment, the content of dsRNA4 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In another preferred embodiment, the content of dsRNA5 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In another preferred embodiment, the content of dsRNA6 in thepharmaceutical composition is 1-500 ng/μl, preferably 5-300 ng/μl, morepreferably 50-150 ng/μl.

In a sixth aspect of the present invention, it provides a use of thedsRNA construct according to the first aspect of the present invention,or the dsRNA according to the second aspect of the present invention, orthe host cell according to the fourth aspect of the present invention,or the composition according to the fifth aspect of the presentinvention, which is selected from the group consisting of:

(1) improving the control effect of aphids; and/or

(2) increasing the dropping rate of insect population; and/or

(3) decreasing the expression level of nymph and/or adult stageregulation-related gene; and/or

(4) reducing the initial number of insect population; and/or

(5) reducing plant damage rate; and/or

(6) reducing crop damage degree and improving the quality of cropproducts.

In a seventh aspect of the present invention, it provides a method forkilling insects, comprising the steps of: using an interference moleculethat interferes with the expression of an insect nymph and/or adultstage regulation-related gene, or feeding or spraying an insect with avector, cell, plant tissue or insect prevention and control reagentcontaining the interference molecule;

preferably, the insect nymph and/or adult stage regulation-related geneis selected from the group consisting of DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, DS45 gene, and a combination thereof.

In another preferred embodiment, the killing insects includes:

(1) improving the control effect of aphids; and/or

(2) increasing the dropping rate of insect population; and/or

(3) decreasing the expression level of nymph and/or adult stageregulation-related gene; and/or

(4) reducing the initial number of insect population; and/or

(5) reducing plant damage rate; and/or

(6) reducing crop damage degree and improving the quality of cropproducts.

In another preferred embodiment, the interference molecule is selectedfrom: dsRNA, antisense nucleic acid, small interfering RNA, and microRNAthat use an insect nymph and/or adult stage regulation-related gene or afragment thereof or a transcript thereof as a target for inhibiting orsilencing.

In another preferred embodiment, the insect nymph and/or adult stageregulation-related gene is derived from the Aphis.

In another preferred embodiment, the insect is a phytophagous insect,preferably from a Hemiptera insect, and most preferably from the Aphis.

In another preferred embodiment, the method includes the steps of: usingthe dsRNA construct according to the first aspect of the presentinvention, or the dsRNA according to the second aspect of the presentinvention, or the host cell according to the fourth aspect of thepresent invention, or the composition according to the fifth aspect ofthe present invention to feed or spray insects.

In an eighth aspect of the present invention, it provides a method forpreparing the dsRNA according to the second aspect of the presentinvention, comprising the steps:

(i) preparing a construct expressing dsRNA, and the construct isdouble-stranded, and its positive or negative strand contains astructure as shown in Formula I:

Seq_(forward)-X-Seq_(reverse)  Formula I

wherein

Seq_(forward) is a nucleotide sequence of insect nymph and/or adultstage regulation-related gene or fragment;

Seq_(reverse) is a nucleotide sequence that is basically complementaryto Seq_(forward);

X is an intervening sequence located between the Seq_(forward) and theSeq_(reverse), and the intervening sequence is not complementary to theSeq_(forward) and the Seq_(reverse),

wherein the insect nymph and/or adult stage regulation-related gene isselected from the group consisting of DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, DS45 gene and a combination thereof;

(ii) transforming the construct as described in step (i) into a hostcell, thereby expressing and forming a dsRNA as shown in Formula II inthe host cell,

wherein

Seq′_(forward) is a RNA sequence or sequence fragment corresponding tothe Seq_(forward) sequence;

Seq′_(reverse) is a sequence that is basically complementary to theSeq′_(forward);

X′ is none; or is an intervening sequence located between Seq′_(forward)and Seq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse),

∥ represents the hydrogen bond formed between Seq_(forward) andSeq_(reverse).

In a ninth aspect of the present invention, it provides a method forpreparing an insect prevention and control reagent comprising the stepsof: spraying the dsRNA construct according to the first aspect of thepresent invention, or the dsRNA according to the second aspect of thepresent invention, or the host cell according to the fourth aspect ofthe present invention, or the composition according to the fifth aspectof the present invention on the surface of the plant, thereby producingthe insect prevention and control agent.

In another preferred embodiment, the plant is selected from the groupconsisting of soybean, radish, peach tree, tobacco, and a combinationthereof.

In a tenth aspect of the present invention, it provides a method forimproving a plant resistance to an insect, comprising:

expressing a recombinant DNA construct in a plant, wherein therecombinant DNA construct comprises DNA encoding RNA, and the RNA has asequence that is substantially identical or substantially complementaryto at least 21 or more consecutive nucleotides of the target gene,wherein the target gene is an insect nymph and/or adult stageregulation-related gene, selected from the group consisting of DS7 gene,DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene, and a combinationthereof.

In another preferred embodiment, the target gene is selected from thegroup consisting of:

(i) a polynucleotide whose sequence is shown in any one of SEQ ID NO.1-6 and 24-29;

(ii) a polynucleotide whose nucleotide sequence is ≥80%, preferably85%-90%, more preferably, 95%, 96%, 97%, 98%, 99% or 100% homologous tothe sequence as shown in any one of SEQ ID NO. 1-6 and 24-29;

(iii) a polynucleotide in which 1-60 (preferably 1-30, more preferably1-10) nucleotides are truncated or added to the 5′end and/or 3′end ofthe polynucleotide as shown in any one of SEQ ID NO. 1-6 and 24-29;

(iv) a polynucleotide which is complementary to any one of thepolynucleotides as described in (i) to (iii).

In another preferred embodiment, the target gene is as shown in any oneof SEQ ID NO. 1-6 and 24-29.

In another preferred embodiment, the homology with the RNA is at least80%, preferably, 85%-100%, more preferably, 95-100%.

In another preferred embodiment, for the DS7 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-1350nt, preferably 506nt-1093nt consecutive nucleotides ofthe target gene.

In another preferred embodiment, for the DS9 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-909nt, preferably 54nt-631nt consecutive nucleotides of thetarget gene.

In another preferred embodiment, for the DS15 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-2148nt, preferably 516nt-1029nt consecutive nucleotides ofthe target gene.

In another preferred example, for the DS25 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-1233nt, preferably 58nt-674nt consecutive nucleotides of the targetgene.

In another preferred example, for the DS27 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-1152nt, preferably 219nt-748nt consecutive nucleotides of thetarget gene.

In another preferred example, for the DS45 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-909nt, preferably 42nt-637nt consecutive nucleotides of the targetgene.

In another preferred embodiment, the RNA is a dsRNA containing at leastone RNA strand.

In another preferred embodiment, the RNA strand includes a sequencehaving at least 90%, preferably 95-100% homology with any one of asequence selected from the group consisting of SEQ ID NO. 1-6 and 24-29.

In another preferred embodiment, the recombinant DNA construct containsa promoter, preferably a heterologous promoter.

In another preferred embodiment, the promoter is selected from the groupconsisting of a constitutive promoter, a space-specific promoter, atime-specific promoter, a development-specific promoter, an induciblepromoter, or a combination thereof.

In another preferred embodiment, the promoter is a promoter that isfunctional in a plant.

In another preferred embodiment, the promoter is selected from the groupconsisting of pol II promoter, pol III promoter, pol IV promoter, pol Vpromoter, and a combination thereof.

In another preferred embodiment, the recombinant DNA construct furthercomprises one or more other elements selected from the group consistingof enhancers, small RNA recognition sites, aptamers or ribozymes,terminators, and additional and extra expression cassettes forexpressing coding sequences (for example, expressing transgenes, such asinsecticidal proteins or selectable markers), non-coding sequences (forexample, expressing additional inhibitory elements), and a combinationthereof.

In another preferred embodiment, the plant further expresses one or moreinsecticidal proteins selected from the group consisting of patatin,phytolectin, plant steroid, Bacillus thuringiensis insecticidal protein,Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein,Bacillus late blight insecticidal protein, and Bacillus sphaericusinsecticidal protein.

In another preferred embodiment, the plant includes a angiosperm and agymnosperm.

In another preferred embodiment, the gymnosperm is selected from thegroup consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae,Taxodiaceae, Cupressaceae, Cephalotaxaceae, Taxaceae, Ephedraceae,Gnetaceae, monotypic family, Welwitschiaceae, and a combination thereof.

In another preferred embodiment, the plant includes monocotyledonousplants and dicotyledonous plants.

In another preferred embodiment, the plant includes a herbaceous plantand a woody plant.

In another preferred embodiment, the herbaceous plant is selected fromthe group consisting of Solanaceae, a gramineous plant, a leguminousplant, and a combination thereof.

In another preferred embodiment, the woody plant is selected from thegroup consisting of Actinidiaceae, Rosaceae, Moraceae, and a combinationthereof.

In another preferred embodiment, the plant is selected from the groupconsisting of a cruciferous plant, a gramineous plant, a leguminousplant, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae,Liliaceae, and a combination thereof.

In another preferred embodiment, the plant is selected from the groupconsisting of Arabidopsis thaliana, Oryza sativa, Chinese cabbage,soybean, tomato, corn, tobacco, wheat, sorghum, radish, and acombination thereof.

In an eleventh aspect of the present invention, it provides a method forpreparing a transgenic plant cell, comprising the steps:

(i) introducing or transfecting a recombinant DNA construct into a plantcell so that the plant cell contains the construct, thereby producingthe transgenic plant cell, wherein the recombinant DNA constructcontains DNA encoding RNA, the RNA has a sequence that is substantiallyidentical or substantially complementary to at least 21 or moreconsecutive nucleotides of the target gene, wherein the target gene isan insect nymph and/or adult stage regulation-related gene, selectedfrom the group consisting of DS7 Gene, DS9 gene, DS15 gene, DS25 gene,DS27 gene, DS45 gene, and a combination thereof.

In another preferred embodiment, the target gene is selected from thegroup consisting of:

(i) a polynucleotide whose sequence is shown in any one of SEQ ID NO.1-6 and 24-29;

(ii) a polynucleotide whose nucleotide sequence is ≥80%, preferably85%-90%, more preferably, 95%, 96%, 97%, 98%, 99% or 100% homologous tothe sequence as shown in any one of SEQ ID NO. 1-6 and 24-29; (Pleasereview)

(iii) a polynucleotide in which 1-60 (preferably 1-30, more preferably1-10) nucleotides are truncated or added to the 5′end and/or 3′end ofthe polynucleotide as shown in any one of SEQ ID NO. 1-6 and 24-29;

(iv) a polynucleotide which is complementary to any of thepolynucleotides as described in (i) to (iii).

In another preferred embodiment, the homology with the RNA is at least80%, preferably, 85%-100%, more preferably, 95-100%.

In another preferred embodiment, for the DS7 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-1350nt, preferably 506nt-1093nt consecutive nucleotides ofthe target gene.

In another preferred embodiment, for the DS9 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-909nt, preferably 54nt-631nt consecutive nucleotides of thetarget gene.

In another preferred embodiment, for the DS15 gene, the RNA has asequence that is substantially identical or substantially complementaryto the 21nt-2148nt, preferably 516nt-1029nt consecutive nucleotides ofthe target gene.

In another preferred example, for the DS25 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-1233nt, preferably 58nt-674nt consecutive nucleotides of the targetgene.

In another preferred example, for the DS27 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-1152nt, preferably 219nt-748nt consecutive nucleotides of thetarget gene.

In another preferred example, for the DS45 gene, the RNA has a sequencethat is substantially identical or substantially complementary to the21nt-909nt, preferably 42nt-637nt consecutive nucleotides of the targetgene.

In another preferred embodiment, the transfection adopts theAgrobacterium transformation method or the gene gun bombardment method.

In a twelfth aspect of the present invention, it provides a method forpreparing a transgenic plant, comprising the steps:

regenerating a transgenic plant cell prepared by the method according tothe eleventh aspect of the present invention into a plant body, therebyobtaining the transgenic plant.

In a thirteenth aspect of the present invention, it provides atransgenic plant cell prepared by the method according to the eleventhaspect of the present invention.

In a fourteenth aspect of the present invention, it provides atransgenic plant prepared by the method according to the twelfth aspectof the present invention.

It should be understood that, within the scope of the present invention,the technical features specifically described above and below (such asthe Examples) can be combined with each other, thereby constituting anew or preferred technical solution which needs not be described one byone limited to the length.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the control effect of target genes on aphids.

FIG. 2 shows the detection results of the relative expression levels oftarget genes.

FIG. 3 shows the control effect of the three target genes of Myzuspersicae in the field.

FIG. 4 shows the results of statistical analysis of the field controleffect and the dropping rate of insect of Myzus persicae.

DETAILED DESCRIPTION OF INVENTION

After extensive and intensive research, the inventors have screened theaphid nymph and/or adult stage regulation-related gene fragments, andunexpectedly found that for the DS7 gene as shown in SEQ ID NO. 1 or 24,the DS9 gene as shown in SEQ ID NO. 2 or 25, the DS15 gene as shown inSEQ ID NO. 3 or 26, DS25 gene as shown in SEQ ID NO. 4 or 27, DS27 geneas shown in SEQ ID NO. 5 or 28, DS45 gene as shown in SEQ ID NO. 6 or29, interfering RNA (dsRNA) is synthesized, and the dsRNA is fed byphytophagous insects (such as Aphis) or directly sprayed on the surfaceof the phytophagous insects, thereby interfering with target genes,inhibiting the expression of target genes, and finally killing aphids.The present invention can also construct plants that can improve insectresistance, and the method of the present invention can also effectivelykill aphids, the control effect of aphids is ≥80%, and the dropping rateof insect is ≥70%. On this basis, the present inventor has completed thepresent invention.

Terms

As used herein, the “crop” refers to various plants cultivated inagriculture, including food crops, economic crop (oil crops, vegetablecrops, flowers, grasses, trees), industrial crops, feed crops, herbcrops, etc., and can grow into large quantities or harvest large areasfor profit or provisions (such as cereal, vegetables, cotton, flax,etc.).

Among them, food crops are mainly rice, corn, beans, potatoes, highlandbarley, broad beans and wheat; oil crops are mainly oilseeds, vines, bigmustard, peanuts, flax, hemp, sunflower, etc.; Vegetable crops mainlyinclude radishes, Chinese cabbage, celery, leeks, garlic, Green onions,carrots, Cucumis melo var flexuosus, lotus vegetables, Jerusalemartichokes, sword bean, coriander, asparagus lettuce, citron day-lily,peppers, cucumbers, tomatoes, coriander, etc.; fruits include pears,green plums, apples, peaches, Apricots, walnuts, plums, cherries,strawberries, crabapple, red dates and other varieties; wild fruitsinclude Pyrus ussuriensis, Armeniaca vulgaris Lam, wild peach, Ziziphusjujuba var. spinosa, prunus maackii, sea-buckthorn, etc.; feed crops aresuch as corn, green manure, Astragalus sinicus, etc.; medicinal cropsare ginseng, Angelica sinensis, Lonicera japonica, mint, mugwort, etc.

RNA Interference (RNAi)

As used herein, the term “RNA interfering (RNAi)” refers to: some smalldouble-stranded RNA can efficiently and specifically block theexpression of specific genes in vivo, promote mRNA degradation, andinduce cells to show a phenotype with a specific gene deletion, which isalso called RNA intervention or RNA interference. RNA interference is ahighly specific gene silencing mechanism at the mRNA level.

As used herein, the term “small interfering RNA (siRNA)” refers to ashort double-stranded RNA molecule that can target mRNA with homologouscomplementary sequences to degrade specific mRNA. This process is theRNA interference pathway.

In the present invention, the basic principle of RNA interference is:using plants as a medium to make insects eat small interfering RNA(siRNA) that can interfere with their gene (such as DS7 genes, DS9genes, DS15 genes, DS25 genes, DS27 genes, DS45 genes) expression,thereby inhibiting the growth of insects.

Specifically, the principle is: through aphids' herbivorous feeding orspraying interfering substances on aphids, RNAi enters the insect bodyand interferes with the RNA of the target gene and inhibits theexpression of the target gene, thereby interfering with the normalgrowth and development of the insect, causing the death of aphids.

As a preferred way, an intron sequence is used to connect complementarygene sequences at both ends. After being introduced into the cell, a“neck-loop” structure can be produced, and the “neck”-shaped part can beprocessed into small RNAs of about 21-25nt in the insect body, which caneffectively inhibit the expression of target genes.

As another preferred way, using the T7 primers in Table 1 to amplifyrespectively, the double-stranded RNA is formed by complementarytranscription, and this double-stranded RNA can be directly used toinhibit the expression of the target gene.

Insect Gene

As used herein, the term “insect gene” refers to a gene related toinsect nymph and/or adult stage regulation. In a preferred embodiment ofthe present invention, the insect gene is DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, and/or DS45 gene. Low or non-expression of thegene will cause abnormalities in the growth, development, metabolism,reproduction and other processes of the insects, and even lead to thedeath of the insects.

As a preferred mode of the present invention, the length of thepreferred insect gene fragment of the present invention is at least 21bp, such as may be 30 bp, 50 bp, 60 bp, 80 bp, 100 bp, 200 bp, 500 bp,1000 bp or the full length of the gene. When the gene is used in thepresent invention, it can be a full-length gene or a gene fragment.Preferably, the fragment for the DS7 gene is shown in SEQ ID NO: 24, thefragment for the DS9 gene is shown in SEQ ID NO: 25, the fragment forthe DS15 gene is shown in SEQ ID NO. 26, the fragment for the DS25 geneis shown in SEQ ID NO. 27, the fragment for the DS27 gene is shown inSEQ ID NO. 28, the fragment for the DS45 gene is shown in SEQ ID NO. 29.The similarities between these fragments and these genes are 85%-100%,respectively, which can produce the same insecticidal effect.

The present invention also provides dsRNA for the DS50 gene, thesequence of the DS50 gene is shown in SEQ ID NO:23. Compared with DS7gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, and/or DS45 gene, thecontrol effect of DS50 gene is not good, the maximum is only about 23%.

The present invention provides interfering RNA targeting insect nymphand/or adult stage regulation-related genes. Insects can take up theinterfering RNAi by oral administration of plants sprayed with RNAi orexpressing dsRNA constructs or dsRNA, or spraying interfering RNAidirectly on the surface of insects.

The dsRNA construct shown in the present invention is shown in FormulaI, and the dsRNA is shown in Formula II. The length of the interveningsequence X used is not particularly limited, as long as it forms aconstruct with the forward sequence and the reverse sequence and whenintroduced into the body, it can form a dsRNA represented by Formula II.As a preferred mode of the present invention, the length of theintervening sequence of the present invention is 80-300 bp; morepreferably 100-250 bp.

In a preferred embodiment of the present invention, the construct forexpressing insect gene dsRNA is introduced into a host cell. The hostcell can be a plant cell, tissue or organ, and the construct can expressan insect gene dsRNA in a plant, and dsRNA is processed into siRNA.Generally, the length of siRNA is about 21-25 nt.

Usually, the construct is located on an expression vector. Theexpression vector usually also contains a promoter, an origin ofreplication, and/or a marker gene, etc. Methods well known to thoseskilled in the art can be used to construct the expression vectorrequired by the present invention. These methods include in vitrorecombinant DNA technology, DNA synthesis technology, and in vivorecombination technology, etc. The expression vector preferably containsone or more selectable marker genes to provide phenotypic traits forselection of transformed host cells, such as kamamycin, gentamicin,hygromycin, and ampicillin resistance.

A vector containing the above-mentioned appropriate gene sequence andappropriate promoter or control sequence can be used to transform anappropriate host. In the method of the present invention, the host maybe any host suitable for carrying the expression vector and capable ofdelivering the expression vector to plant cells. Preferably, the host isAgrobacterium.

Although the insects exemplified in the examples of the presentinvention are aphids. However, it should be understood that the presentinvention has no particular limitations on the insects applicable to thepresent invention. The insects may be any phytophagous insects that canfeed on plants, for example, they may be Hemiptera insects.

The present invention has no particular limitations on the plantsapplicable to the present invention. Plants eaten by aphids arepreferred, such as soybeans, radishes, peach trees, tobacco and thelike.

DS7 Gene

As used herein, the terms “DS7 gene”, “tubulin alpha chain-like”, and“tubulin a chain” can be used interchangeably, and they are all widelydistributed globular proteins and are the basic structural unit ofmicrotubules in cells. It plays an important role in cell movement anddivision, and is expressed in the nymph stage.

In the present invention, some glutamic acid residues at the C-terminusof the protein are polyglutamylated, resulting in a polyglutamic acidchain on the γ-carboxyl group. Polyglutamylation plays a key role inspastin (SPAST) microtubule cutting. SPAST preferentially recognizes andacts on microtubules modified with short polyglutamic acid tails: thecleavage activity of SPAST increases as the number of glutamate pertubulin increasing from 1 to 8, but the decrease exceeds theglutamylation threshold.

Some glutamic acid residues at the C-terminus are monoglycosylated, butnot polyglycerinated. Monoglycination is mainly limited to tubulin(cilia and flagella) incorporated into axoneme. Both polypentanoylationand monoglycination can coexist on the same protein on adjacentresidues, and reducing the level of glycylation can increasepolypentanoylation and interact with each other.

In one embodiment of the present invention, based on RNAi technology,the DS7 gene is used as a target to screen interfering RNA fragmentsagainst the DS7 gene. Preferably, the sequence of the DS7 gene fragmentis shown in SEQ ID NO:1 or 24:

(SEQ ID NO.: 1) ATGCGTGAATGTATCTCTGTACACGTTGGCCAAGCTGGTGTTCAAATCGGTAATGCCTGCTGGGAATTGTACTGTTTGGAACATGGAATTGCTCCAGATGGTCAAATGCCATCTGACAAGACCATTGGAGGTGGAGACGACAGCTTCAACACCTTCTTCAGCGAAACTGGCTCAGGCAAACATGTGCCAAGAGCTGTGTTCGTTGATCTCGAACCAACTGTTGTTGATGAGGTAAGAACTGGAACATACCGCCAGTTGTTCCACCCTGAACAATTGATCACTGGTAAGGAAGATGCCGCCAACAACTACGCACGTGGACACTACACTATCGGAAAAGAGATTGTTGATGTTGTTTTGGACCGAATCAGGAAATTGGCTGATCAGTGCACTGGTCTTCAAGGTTTCCTGATCTTCCACTCTTTCGGAGGTGGTACTGGATCTGGTTTCACATCTTTGTTGATGGAAAGACTCAGCGTTGACTACGGAAAGAAGAGTAAATTAGAATTCGCCATCTACCCAGCCCCTCAAGTATCCACAGCTGTAGTTGAGCCATACAACTCCATCTTGACCACACATACAACTCTTGAACACAGTGACTGTGCATTCATGGTCGATAATGAAGCCATCTATGACATCTGCCGTCGTAATCTCGATATTGAACGTCCAACTTACACTAACTTGAATCGTCTTATTGGCCAGATTGTTTCTTCAATCACAGCTTCTCTCCGTTTCGATGGTGCCCTCAATGTTGACTTGACTGAATTCCAGACCAATTTGGTCCCATACCCCCGTATTCATTTCCCATTGGTCACCTATGCACCAGTCATCTCCGCTGAAAAGGCTTACCATGAACAATTGTCCGTATCAGAAATCACTAACGCTTGTTTTGAACCAGCCAACCAAATGGTGAAATGTGATCCACGTCATGGCAAATACATGGCTTGTTGCATGTTGTACCGTGGTGATGTTGTACCCAAAGACGTCAACGCTGCCATTGCTTCCATCAAGACCAAGAGAACAATTCAGTTTGTTGACTGGTGTCCAACTGGTTTCAAAGTTGGTATCAACTACCAACCCCCAACCGTGGTACCCGGTGGTGACTTGGCTAAGGTACAACGTGCCGTCTGCATGTTGTCCAACACTACAGCTATTGCTGAAGCTTGGGCTAGGTTGGACCACAAGTTCGACTTGATGTACGCCAAACGTGCTTTCGTCCATTGGTATGTTGGAGAAGGTATGGAAGAAGGAGAATTCTCTGAAGCTCGTGAGGATTTGGCTGCTCTAGAGAAAGATTACGAAGAGGTTGGCATGGACTCCGTCGAAGGCGAAGGCGAAGGTGGTGAAGAATA C (SEQ ID NO.: 24)TAATACGACTCACTATAGGGAGATCGCCATCTACCCAGCCCCTCAAGTATCCACAGCTGTAGTTGAGCCATACAACTCCATCTTGACCACACATACAACTCTTGAACACAGTGACTGTGCATTCATGGTCGATAATGAAGCCATCTATGACATCTGCCGTCGTAATCTCGATATTGAACGTCCAACTTACACTAACTTGAATCGTCTTATTGGCCAGATTGTTTCTTCAATCACAGCTTCTCTCCGTTTCGATGGTGCCCTCAATGTTGACTTGACTGAATTCCAGACCAATTTGGTCCCATACCCCCGTATTCATTTCCCATTGGTCACCTATGCACCAGTCATCTCCGCTGAAAAGGCTTACCATGAACAATTGTCCGTATCAGAAATCACTAACGCTTGTTTTGAACCAGCCAACCAAATGGTGAAATGTGATCCACGTCATGGCAAATACATGGCTTGTTGCATGTTGTACCGTGGTGATGTTGTACCCAAAGACGTCAACGCTGCCATTGCTTCCATCAAGACCAAGAGAACAATTCAGTTTGTTGACTGGTGTCCAACTGGTTTCAAAGTTGGTATCAACTACCAACCCCCAACCGTGGTACCCGAGAGGGATATCACTCAGCATAAT

DS9 gene

As used herein, the terms “DS9 gene”, “ADP/ATP translocase 3-like”, and“ADP/ATP carrier protein (AAC)” can be used interchangeably, and areresponsible for transporting phosphorylated synthesis of ATP to thecytoplasm as the main ability supply of cells, providing power forthermodynamic reaction, which is expressed in the nymph stage.

In the present invention, this protein is a transport protein thatallows the intracellular exchange of adenosine diphosphate (ADP) andmitochondrial adenosine triphosphate (ATP) to cross the mitochondrialinner membrane. Free ADP is transported from the cytoplasm to themitochondrial matrix, while ATP produced by oxidative phosphorylation istransported from the mitochondrial matrix to the cytoplasm, therebyproviding the cell with the main energy.

In one embodiment of the present invention, based on the RNAitechnology, the DS9 gene is used as a target to screen the RNA fragmentsagainst the DS9 gene. Preferably, the sequence of the DS9 gene fragmentis shown in SEQ ID NO: 2 or 25:

(SEQ ID NO.: 2) ATGGCCGAAACCAAAGCGCCGAAGGACCCGTATGGTTTCTTGAAGGACTTCATGGCCGGTGGTATCTCCGCTGCCGTGTCGAAGACCGCCGTGGCTCCGATCGAGCGCGTCAAGCTTATCCTGCAAGTGCAGGCCGCTTCCACGCAGATCGCCGCCGACCAACAGTACAAAGGAATTATGGACTGTTTGGTGAGAATCCCAAAAGAACAAGGATTTGCCAGTTTCTGGAGAGGTAACTTTGCCAATGTCATCAGGTACTTCCCAACACAAGCATTGAACTTTGCTTTCAAGGATGTCTACAAACAGGTGTTTATGGACGGTGTGGATAAAAAGACTCAATTCTGGCGGTATTTTGCTGGTAACTTGGCATCTGGTGGTGCTGCTGGAGCAACATCTTTGTGCTTTGTATACCCCCTCGATTACGCACGTACACGATTAGGAGCTGATGTCGGTAAAGGACCAGCTGAAAGGCAGTTCAAAGGTCTTGGTGATTGTTTAGCCAAAACCGTCAAGTCTGATGGTCCCATTGGTTTGTACCGTGGTTTCATTGTATCAGTACAGGGTATCATCATCTACCGTGCTGCATACTTTGGATTTTTCGACACAGCTAAGGGAATGTTGCCAGACCCCAAGAATACTCCATTCTTAGTTTCATGGGGTATCGCCCAATTTGTAACAACATTCGCTGGTATTATGTCCTATCCATTTGACACAGTCAGACGTCGTATGATGATGCAATCTGGCCGTGCTGCTGACCAACGCATGTACAAGAGCACATTGGACTGCTGGGGTAAACTTTACAAGAATGAAGGTACATCTGCTTTCTTCAAGGGTGCATTCTCCAACGTACTCAGAGGTACTGGTGGTGCCTTGGTGTTGGTCTTCTACGACGAACTCA AAAACCTCATG  (SEQ ID NO.: 25) TAATACGACTCACTATAGGGAGAGCCGGTGGTATCTCCGCTGCCGTGTCGAAGACCGCCGTGGCTCCGATCGAGCGCGTCAAGCTTATCCTGCAAGTGCAGGCCGCTTCCACGCAGATCGCCGCCGACCAACAGTACAAAGGAATTATGGACTGTTTGGTGAGAATCCCAAAAGAACAAGGATTTGCCAGTTTCTGGAGAGGTAACTTTGCCAATGTCATCAGGTACTTCCCAACACAAGCATTGAACTTTGCTTTCAAGGATGTCTACAAACAGGTGTTTATGGACGGTGTGGATAAAAAGACTCAATTCTGGCGGTATTTTGCTGGTAACTTGGCATCTGGTGGTGCTGCTGGAGCAACATCTTTGTGCTTTGTATACCCCCTCGATTACGCACGTACACGATTAGGAGCTGATGTCGGTAAAGGACCAGCTGAAAGGCAGTTCAAAGGTCTTGGTGATTGTTTAGCCAAAACCGTCAAGTCTGATGGTCCCATTGGTTTGTACCGTGGTTTCATTGTATCAGTACAGGGTATCATCATCTACCGTGCTGCATACTTTGGATTTTTCGACACAGCTAAGGGAATGTTGCCAGACCCCA AGAGGGATATCACTCAGCATAAT

DS15 Gene

As used herein, the terms “DS15 gene”, “heat shock protein 83-like”, and“heat shock protein 83” can be used interchangeably. They areintracellular molecular chaperone proteins that play an important rolein protein interactions, such as assisting in folding and assisting inthe establishment of a suitable protein conformation, which is expressedin the nymph stage.

In the present invention, heat shock proteins (HSP) are a family ofproteins produced by cells in response to exposure to stress conditions.They are first associated with heat shock, but are now known in otherstresses, including exposure to cold, and in wound healing or tissueremodeling. Many members of this group perform chaperone molecularfunctions by stabilizing new proteins to ensure proper folding or byhelping to fold proteins damaged by cellular stress. Increasement is theregulation of transcription. The significant up-regulation of heat shockproteins is a key part of the heat shock response, which is mainlyinduced by heat shock factor (HSF).

In one embodiment of the present invention, based on RNAi technology,the DS15 gene is used as a target to screen RNA fragments against theDS15 gene. Preferably, the sequence of the DS15 gene fragment is shownin SEQ ID NO: 3 or 26:

(SEQ ID NO.: 3) ATGCCTGAAGACGTTACCATGACTGCATCTGATGATGTTGAGACCTTCGCTTTCCAAGCTGAGATCGCTCAGCTTATGTCCCTCATCATCAACACCTTCTACTCGAACAAAGAAATCTTTTTGCGAGAATTGGTATCCAATTCTTCTGATGCATTGGACAAAATTCGTTATGAGTCATTGACTGATCCATCCAAATTGGAATCTGGCAAAGATTTACACATTAAAATCATCCCCAATGCGGAAGAAAAAACTCTGACCATTATTGACACTGGTATCGGTATGACCAAAGCTGATCTAGTCAACAACTTGGGAACCATTGCTAAATCTGGTACTAAGGCTTTCATGGAAGCTTTACAAGCTGGAGCTGATATTTCCATGATTGGTCAATTTGGTGTGGGTTTCTATTCCGCCTATCTGGTAGCTGACAAAGTCACTGTTGTTTCCAAACACAACGACGATGAACAATATTTGTGGGAATCTGCTGCCGGAGGTTCATTCACCATCCGTACTGATCCTGGTGAACCATTGGGCCGTGGTACCAAAATTGTCCTTCAAATCAAAGAAGATCAAGCTGAGTTCCTCCAACAAGAAAAAATTACCAGCATCATCAAGAAGCACTCTCAATTCATTGGCTACCCAATCAAATTAATCGTTGAGAATGAACGTACCAAAGAAGTCAGCGATGATGAAGCTGAAGAAGAAAAGAAAGATGAAGTTGAAGGTGAAACTGAAGAAGACAAAAAACCCAAAATTGAGGATGTTGGTGAGGATGAAGACGAAGACAAAAAAGATGAAGACAAAGACAAAAAGAAGAAGAAGACTATTAAAGAAAAGTACTTGGATGAAGAGGTCTTGAACAAGACAAAACCAATCTGGACACGCAACCCTGATGATATCAGCCAAGATGAATATGGTGAATTCTACAAATCCTTAACCAATGACTGGGAAGATCATTTAGCCGTCAAACATTTCTCTGTGGAAGGACAACTTGAATTCAGAGCATTGTTATTCATTCCCAAGCGTGCGCCTTATGACATGTTTGAGAACAAGAAGAAGAAGAACAACATTAAATTATATGTCCGTCGTGTCTTCATCATGGACAACTGCGAAGACCTCATGCCAGAATACTTGAACTTCATCAAGGGTGTTGTTGACAGTGAGGATTTGCCGTTGAACATCTCCCGTGAAATGCTCCAACAAAACAAGATCTTGAAAGTTATCAGGAAGAATTTGGTTAAGAAATGTTTGGAATTGTTCGAGGAATTGGCTGAAGACAAGGACAACTACAAGAAATTGTACGAACAGTTCAGCAAGAACTTGAAACTTGGAATCCACGAAGATAGCCAAAACAGAAAGAAACTCTCAGACTTGTTGAGATTCCACTCCTCAGCCAGTGGTGACGAATCATGCTCCCTTAAGGAGTATGTTGCACGTATGAAGCCAAATCAAACCCACATTTACTACATCACAGGTGAAAGCCGTGAACAAGTATCCAACTCTTCATTCGTTGAACGTGTCAAGAAACGTGGTTTTGAAGTTATTTACATGACTGAACCCATTGATGAATACGTTGTCCAACAAATGAAAGAATATGACGGCAAGAACTTGGTATCTGTCACTAAAGAAGGTTTGGACTTGCCTGAAACCGATGAAGAAAAGAAGAAGCGCGAGGATGATCAATCCAGATTTGAAAAATTGTGCAAAGTTGTTAAGGACATTTTGGACAAGAAAGTTGAGAAGGTTGTCATCAGTAACAGACTTGTTGAGTCTCCCTGTTGCATTGTCACATCTCAGTATGGTTGGACTGCCAACATGGAACGTATCATGAAGGCACAAGCACTCAGAGATTCATCTACCATGGGTTATATGTCTGCCAAAAAACACTTGGAAATCAACCCTGACCACCCGATCATTGAAACACTCAGACAAAAGGCTGAAGCTGATTGCAACGACAAGGCTGTCAGAGACTTGGTCATGCTTTTGTTCGAGACAAGTTTGTTGTCATCTGGTTTTGGACTTGAAGACCCACAAGTTCACGCTTCTAGAATCCACAGAATGATCAAATTGGGTTTGGGCATTGATGAAGATTTGCCAGTAGTTGAAGAAAAATCTGCTGAAGTTGAAGCCTCCGAGCCTGTTGTTGAAGCTGATGCTGAAGATTCTTCTCGCATGGAAGAAGTTGAT (SEQ ID NO.: 26)TAATACGACTCACTATAGGGAGATGGTGAACCATTGGGCCGTGGTACCAAAATTGTCCTTCAAATCAAAGAAGATCAAGCTGAGTTCCTCCAACAAGAAAAAATTACCAGCATCATCAAGAAGCACTCTCAATTCATTGGCTACCCAATCAAATTAATCGTTGAGAATGAACGTACCAAAGAAGTCAGCGATGATGAAGCTGAAGAAGAAAAGAAAGATGAAGTTGAAGGTGAAACTGAAGAAGACAAAAAACCCAAAATTGAGGATGTTGGTGAGGATGAAGACGAAGACAAAAAAGATGAAGACAAAGACAAAAAGAAGAAGAAGACTATTAAAGAAAAGTACTTGGATGAAGAGGTCTTGAACAAGACAAAACCAATCTGGACACGCAACCCTGATGATATCAGCCAAGATGAATATGGTGAATTCTACAAATCCTTAACCAATGACTGGGAAGATCATTTAGCCGTCAAACATTTCTCTGTGGAAGGACAACTTGAATTCAGAGCATTGTTATTCATTCCCAAGCGTGCGCCT AGAGGGATATCACTCAGCATAAT

DS25 Gene

As used herein, the terms “DS25 gene”, “eukaryotic initiation factor4A-like”, and “eukaryotic initiation factor complex type of 4A” can beused interchangeably, it is a helicase that unwinds double-stranded RNAand is also a functional protein necessary for ribosomal subunitbinding, which is expressed in the nymph stage.

In the present invention, the eukaryotic initiation factor complex formsa ternary complex with GTP and the initiator Met-tRNA. This process isregulated by guanine nucleotide exchange and phosphorylation, and is themain regulatory element of the bottleneck of gene expression. Before thetranslation progresses to the extension stage, many initiation factorsmust promote the synergy of ribosomes and mRNA, and ensure that the5′UTR of the mRNA is sufficiently lacking in secondary structure. Thefourth group of eukaryotic initiation factors promotes this combination;it is of significance in the normal regulation of translation and thetransformation and progression of cancer cells.

In one embodiment of the present invention, based on RNAi technology,the DS25 gene is used as a target to screen RNA fragments against theDS25 gene. Preferably, the sequence of the DS25 gene fragment is shownin SEQ ID NO: 4 or 27:

(SEQ ID NO.: 4) ATGAATGCTAATGAGACGAAAAATGGACCTCCTAGTGAAACCAATGACTACTCGGGACCACCTGGCATGGACGTCGGTGGAACTATTGAGTCTGACTGGAAAGAAGTGGTGGATAACTTTGATGAGATGAATTTAAAAGAAGAATTGTTGCGTGGTATTTATGGATATGGTTTTGAAAAGCCATCAGCTATTCAACAACGTGCTATTTTGCCGTGCATCAAGGGACATGATGTCATTGCTCAGGCCCAATCTGGTACTGGCAAGACAGCTACTTTTTCCATTTCTATTCTCCAACAAATTGATACAAGTTTGAATGAGTGCCAAGCACTTATTTTGGCACCAACACGTGAATTGGCTCAACAGATTCAAAAGGTGGTCATTGCTTTGGGTGATTTCATGAAAGCTGATTGTCATGCTTGCATTGGCGGTACAAACGTTCGTGATGACATGCGTAAGCTGGATACTGGATCCCATGTAGTTGTTGGAACTCCTGGCCGTGTTTATGACATGATTGCTAGAAAATCCCTAAGAACTCAATTTATCAAGATATTTGTGTTGGACGAAGCTGATGAAATGTTGTCTCGAGGTTTCAAAGATCAAATTAAAGAGGTGTTCAAGTTCCTCGAAGAAGATATTCAGGTCATTCTGTTGTCTGCTACAATGCCCGAGGACGTTTTGGATGTGAGCACTCATTTCATGCGTAATCCAGTACGCATTCTTGTTCAAAAGGAAGAACTGACATTGGAAGGTATCAAACAGTTTTACATCAATGTTACCAAAGAAGAATGGAAGTTTGACACTCTATGTGATTTGTACGACACTCTTAGTATCACCCAGGCTGTGATCTTCTGTAACACACGTCGTAAGGTAGAGTGGTTGACTGAAAATATGCGTTTGAAAACATTTACTGTATCAGCTATGCATGGAGAAATGGACCAACGTCAACGTGAGCTAATTATGCGTCAATTCCGTTCTGGCTCTAGTCGTGTTCTAATTACCACTGATTTGTTGGCTCGAGGCATTGATGTACAACAAGTTTCTCTGGTCATCAATTACGATTTGCCGTCCAATCGTGAAAACTATATTCACAGGATTGGACGTTCTGGCCGTTTCGGTCGTAAAGGAGTCGCCATTAATTTTATCACCGAAGACGACAAAAGAGCTATGAAGGATATTGAATCATTTTACAACACTCACGTGCTCGAGATGCCACAGAATGTGGCCGATTTGCTG (SEQ ID NO.: 27)TAATACGACTCACTATAGGGAGACCACCTGGCATGGACGTCGGTGGAACTATTGAGTCTGACTGGAAAGAAGTGGTGGATAACTTTGATGAGATGAATTTAAAAGAAGAATTGTTGCGTGGTATTTATGGATATGGTTTTGAAAAGCCATCAGCTATTCAACAACGTGCTATTTTGCCGTGCATCAAGGGACATGATGTCATTGCTCAGGCCCAATCTGGTACTGGCAAGACAGCTACTTTTTCCATTTCTATTCTCCAACAAATTGATACAAGTTTGAATGAGTGCCAAGCACTTATTTTGGCACCAACACGTGAATTGGCTCAACAGATTCAAAAGGTGGTCATTGCTTTGGGTGATTTCATGAAAGCTGATTGTCATGCTTGCATTGGCGGTACAAACGTTCGTGATGACATGCGTAAGCTGGATACTGGATCCCATGTAGTTGTTGGAACTCCTGGCCGTGTTTATGACATGATTGCTAGAAAATCCCTAAGAACTCAATTTATCAAGATATTTGTGTTGGACGAAGCTGATGAAATGTTGTCTCGAGGTTTCAAAGATCAAATTAAAGAGGTGTTCAAGTTCCTCGAAGAAGATATTCAGGTCATTCTGTTGTCTGCTACAATGCCCGAGGACGT AGAGGGATATCACTCAGCATAAT

DS27 Gene

As used herein, the terms “DS27”, “troponin T-like isoform 3”, and“troponin type 3” can be used interchangeably. It mediates Ca ionchannels and regulates the contraction regulation function of insectstriated muscle, which is expressed in the nymph and adult stages.

In the present invention, troponin is attached to the proteintropomyosin and is located in the grooves between actin filaments inmuscle tissue. In a relaxed muscle, tropomyosin blocks the attachmentsite of the myosin cross bridge, thereby preventing contraction. Whenmuscle cells are stimulated to contract by an action potential, calciumchannels open in the sarcoplasm membrane and release calcium into thesarcoplasm. Some of this calcium attaches to troponin, causing it tochange shape, exposing the binding site of myosin (active site) on actinfilaments. The binding of myosin to actin causes cross bridges to formand start to contract muscles.

Troponin activation. Troponin C (red) binds to Ca2⁺ and stabilizes theactivated state, wherein troponin I (yellow) no longer binds to actin.Troponin T (blue) fixes the complex to tropomyosin.

Troponin is found in skeletal muscle and heart muscle, but the specificversion of troponin differs in different types of muscles. The maindifference is that the TnC subunit of troponin has four calcium bindingsites in skeletal muscle, but only three in cardiac muscle. Opinions onthe actual content of calcium bound to troponin vary from expert tosource.

In one embodiment of the present invention, based on the RNAitechnology, the DS27 gene is used as a target to screen the RNAfragments against the DS27 gene. Preferably, the sequence of the DS27gene fragment is shown in SEQ ID NO: 5 or 28:

(SEQ ID NO.: 5) ATGTCCGACGAAGAAGAAGTGTACACTGATTCCGAAGAAGAAACGCAACCGGAGCCTGAAAAAAGCAAAGATGGAGATGGAGATCCCGAATTCGTTAAGAGGCAAGAATTAAAATCTTCAGCCTTAGACGAACAGCTTAAAGAGTACATCCAAGAATGGCGCAAACAGCGGTCAAAGGAAGAAGACGACTTAAAGAAGTTGAAGGAAAAACAGGCCAAGCGCAAGGTTATGCGAGCGGAAGAAGAGAAGAGAATGGCCGAGAGAAAGAAGCAAGAAGAAGAACGCAGACAGAGAGAAGTCGAGGAAAAGAAACAAAAGGACATCGAAGAAAAACGTAAACGTCTAGAAGAGGCCGAGAAAAAACGGCAAGCTATGATGGCTGCTCTTAAGGAACAAACCAATAAATCTAAAGGACCAAATTTCACCATCAGCAAAAAAGAAGGTGCGTTGAGTATGACTTCTGCCCAACTTGAACGCAATAAAACCAGAGAACAGATCGAAGAAGAAAAGAAAATATCGTTGAGCTTCAGAATCAAACCTTTGAATATTGAAGGATTCTCTGTGCAAAAACTCCAATTCAAAGCTACCGAACTCTGGGACCAGATCATCAAGTTGGAAACAGAAAAATACGATTTGGAGGAAAGGCAAAAGAGACAAGATTACGACTTGAAAGAGTTGAAAGAACGTCAGAAGCAACAACTCCGCCACAAGGCTCTGAAGAAAGGTCTCGACCCCGAAGCCCTAACCGGCAAATACCCACCCAAGATCCAAGTCGCTTCCAAGTACGAGAGGCGAGTTGACACGAGGTCTTATGATGACAAAAAGAAGCTGTTCGAAGGAGGTTATATGGAAACCACTAAAGAATCAATGGAAAAACAATGGACAGAAAAAAGTGACCAATTCGGTGGCCGCGCTAAAGGACGATTACCGAAATGGTTCGGCGAACGTCCGGGCAAGAAGAAGGATGACCCAGACACACCCGAAGAGGAAGAGCTCAAGAAAAACGAGGAAGACGAAGAACCGTTTGGCCTCGACGACGAAGAAGCTGAAGAAGAAGTTGAAGAGGAAGAAGAGGAGGAAGAAGAAGAGGAAGAGGAGGAGGAAGAGGAAGAAGAGGAAGAAGAAGAAGAGGAAGAGGAAGAAGAAGAAGAA (SEQ ID NO.: 28)TAATACGACTCACTATAGGGAGAGCGCAAGGTTATGCGAGCGGAAGAAGAGAAGAGAATGGCCGAGAGAAAGAAGCAAGAAGAAGAACGCAGACAGAGAGAAGTCGAGGAAAAGAAACAAAAGGACATCGAAGAAAAACGTAAACGTCTAGAAGAGGCCGAGAAAAAACGGCAAGCTATGATGGCTGCTCTTAAGGAACAAACCAATAAATCTAAAGGACCAAATTTCACCATCAGCAAAAAAGAAGGTGCGTTGAGTATGACTTCTGCCCAACTTGAACGCAATAAAACCAGAGAACAGATCGAAGAAGAAAAGAAAATATCGTTGAGCTTCAGAATCAAACCTTTGAATATTGAAGGATTCTCTGTGCAAAAACTCCAATTCAAAGCTACCGAACTCTGGGACCAGATCATCAAGTTGGAAACAGAAAAATACGATTTGGAGGAAAGGCAAAAGAGACAAGATTACGACTTGAAAGAGTTGAAAGAACGTCAGAAGCAACAACTCCGCCACAAGGCTCTGAAGAAAGGTCTCGACCCCGAAGCCCTAACCGAGAGGGATATCACTCAGCATAAT

DS45 Gene

As used herein, the terms “DS45 gene”, “Y-box protein Ct-p40-like”, and“y box binding protein Ct-p40-like” can be used interchangeably, andaffect cell differentiation and cytoskeleton formation. Deletion of itwill inhibit signal transduction pathways inside and outside the cell,involved in DNA damage repair and transcription, and it is expressed inthe nymph stage.

In one embodiment of the present invention, based on RNAi technology,the DS45 gene is used as a target to screen RNA fragments directedagainst the DS45 gene. Preferably, the sequence of the DS45 genefragment is shown in SEQ ID NO: 6 or 29:

(SEQ ID NO.: 6) ATGGCGGAACAAGTCGGCGAGAGGAGGACGGAACGGCCGCCGCAGAAGCCCGTGGCCCAAAAGCCGGTCATATCTGTGAAAGTCACCGGCGTTGTTAAATGGTTCAACGTCAAAAGCGGTTATGGTTTTATTAATCGTAATGATACAAAAGAAGATATATTTGTACATCAGTCTGCTATTATCAAGAACAACCCTAAGAAAATTGTACGCAGTGTCGGTGATGGAGAAACTGTAGAATTTGACGTTGTTGAGGGCGAAAAAGGTCACGAAGCAGCAAATGTTACTGGTCCAGATGGAGAAGCTGTTAAAGGATCACCTTATGCAGCTGAAAGAAGAAGAAATAACTATCGTCAGTGGTTTTATGGACGCCGTCCTAATACCCGTCCAAGAAATGGTGGTCAACCTCCAAGAGATGGTAGTCCAAGTGGTGACAAGGAAGAAACTGAAAATGAAGTAGGAGAACAACCAAGACGTTACCGCCAGCCACGTCAACAGAATTGGTATAATAGCTATCGTGGAAATCGAAGAGGTCCACCACCAAATAGAGGAGAAGGTGGTGATTACAATGGTGGAGATAATTATGGATATGATAGTTCACCTCCTGGTAGAGGCAGAGGTCGTGGGATGGGTGCGCCTAGACGTTTCTTTAGACGTGGCAGTGGATTTAGAGGGAGCCGTGGAACAGGTGGTCCACCCAGAAGACCATATCAAGATGAAAATCAGGACAATGAATATAATCAAAGTGATGAAAATGGAGCAAATAGACCTCGTCCTCGCTATCGCCGCCGCAATAATCGTTCTAGAGCGAGAAGTGATGGTCCTCCAAGAGCCAATAGCCAAAGTGACAATGAATCTAAACAAAAAAACTTTGGAGGAGAAGCATTGGA ACTGGATGAAAGTAGTCATGCT(SEQ ID NO.: 29) TAATACGACTCACTATAGGGAGAGCAGAAGCCCGTGGCCCAAAAGCCGGTCATATCTGTGAAAGTCACCGGCGTTGTTAAATGGTTCAACGTCAAAAGCGGTTATGGTTTTATTAATCGTAATGATACAAAAGAAGATATATTTGTACATCAGTCTGCTATTATCAAGAACAACCCTAAGAAAATTGTACGCAGTGTCGGTGATGGAGAAACTGTAGAATTTGACGTTGTTGAGGGCGAAAAAGGTCACGAAGCAGCAAATGTTACTGGTCCAGATGGAGAAGCTGTTAAAGGATCACCTTATGCAGCTGAAAGAAGAAGAAATAACTATCGTCAGTGGTTTTATGGACGCCGTCCTAATACCCGTCCAAGAAATGGTGGTCAACCTCCAAGAGATGGTAGTCCAAGTGGTGACAAGGAAGAAACTGAAAATGAAGTAGGAGAACAACCAAGACGTTACCGCCAGCCACGTCAACAGAATTGGTATAATAGCTATCGTGGAAATCGAAGAGGTCCACCACCAAATAGAGGAGAAGGTGGTGATTACAATGGTGGAGATAATTATGGATATGATAGTTCACCTCCTGGTAGAGGCAGAGGTCGTGGGATGGGTGCGCCTAAGAGGGATATCACTCAGCATAA T

dsRNA Construct and its Application

The present invention provides a dsRNA construct. The dsRNA construct isdouble-stranded, and its positive or negative strand contains astructure as shown in Formula I:

Seq_(forward)-X-Seq_(reverse)  Formula I

wherein

Seq_(forward) is a nucleotide sequence of insect nymph and/or adultstage regulation-related gene or fragment;

Seq_(reverse) is a nucleotide sequence that is basically complementaryto Seq_(forward);

X is an intervening sequence located between the Seq_(forward) and theSeq_(reverse), and the intervening sequence is not complementary to theSeq_(forward) and the Seq_(reverse),

wherein the insect nymph and/or adult stage regulation-related gene isselected from the group consisting of DS7 gene, DS9 gene, DS15 gene,DS25 gene, DS27 gene, DS45 gene and a combination thereof.

In a preferred embodiment of the present invention, the length of theSeq_(forward) and Seq_(reverse) is at least 50 bp.

In a preferred embodiment of the present invention, the dsRNA constructis ingested by insects (such as aphids) to form a dsRNA of Formula II,

wherein

Seq′_(forward) is a RNA sequence or sequence fragment corresponding tothe Seq_(forward) sequence;

Seq′_(reverse) is a sequence that is basically complementary to theSeq′_(forward);

X′ is none; or is an intervening sequence located between Seq′_(forward)and Seq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse),

∥ represents the hydrogen bond formed between Seq_(forward) andSeq_(reverse).

The present invention also provides the use of the dsRNA construct,which is used to: (1) improve the control effect of aphids; and/or (2)increase the dropping rate of insect population; and/or (3) reduce theexpression level of nymph and/or adult stage regulation-related gene;(4) reduce the initial number of insect population; and/or (5) reducethe damage rate of plants; and/or (6) reduce the damage degree of cropsand improve the quality of crop products.

dsRNA and its Applications

The present invention also provides a dsRNA as shown in Formula II,

wherein

Seq′_(forward) is a RNA sequence or sequence fragment corresponding toSeq′_(forward) sequence;

Seq′_(reverse) is a sequence that is basically complementary to theSeq′_(forward);

X′ is none; or is an intervening sequence located between Seq′_(forward)and Seq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse);

∥ represents the hydrogen bond formed between Seq_(forward) andSeq_(reverse).

In another preferred embodiment, the length of the intervening sequenceX is 0-300 bp, preferably 100 bp.

The insect nymph and/or adult stage regulation-related gene is derivedfrom aphids; the sequence of the DS7 gene is shown in SEQ ID NO. 1; thesequence of the DS9 gene is shown in SEQ ID NO. 2; The sequence of theDS15 gene is shown in SEQ ID NO. 3; the sequence of the DS25 gene isshown in SEQ ID NO. 4; the sequence of the DS27 gene is shown in SEQ IDNO. 5; the sequence of the DS45 gene is shown in SEQ ID NO. 6.

In another preferred embodiment, the insects are phytophagous insects,preferably from Hemiptera insects, and most preferably from Aphis.

The present invention also provides the use of the dsRNA, which is usedto: (1) improve the control effect of aphids; and/or (2) increase thedropping rate of insect population; and/or (3) reduce the expressionlevel of nymph and/or adult stage regulation-related gene; and/or (4)reduce the initial number of insect population; and/or (5) reduce thedamage rate of plants.

Composition and its Application

The present invention also provides a composition. In response to theproblem of efficiently killing aphids, the inventor has developed RNAifragments for target genes based on RNAi technology, and improved thecontrol effect of aphids and the dropping rate of insect population byfeeding insects or spraying insects directly, making RNAi have theeffect of inhibiting gene expression, and finally achieving the purposeof efficiently killing aphids. The method of the present invention isefficient, convenient, fast, accurate and pollution-free.

The composition includes a dsRNA construct and/or dsRNA, and aneffective amount of a carrier acceptable for insect feeding. In anotherpreferred embodiment, the composition is a composition used to induce orcause the death of aphid nymphs and/or adult stage.

In another preferred embodiment, the dsRNA has the following sequence:

dsRNA1: having a sequence corresponding to SEQ ID NO. 1 or 24;

dsRNA2: having a sequence corresponding to SEQ ID NO. 2 or 25;

dsRNA3: having a sequence corresponding to SEQ ID NO. 3 or 26;

dsRNA4: having a sequence corresponding to SEQ ID NO. 4 or 27;

dsRNA5: having a sequence corresponding to SEQ ID NO. 5 or 28;

dsRNA6: having a sequence corresponding to SEQ ID NO. 6 or 29.

The present invention also provides a use of the composition, which isselected from the following group:

(1) improving the control effect of aphids; and/or

(2) increasing the dropping rate of insect population; and/or

(3) decreasing the expression level of nymph and/or adult stageregulation-related gene; and/or

(4) reducing the initial number of insect population; and/or

(5) reducing plant damage rate; and/or

(6) reducing crop damage degree and improving the quality of cropproducts.

In a preferred embodiment of the present invention, the composition isan aqueous solution, and the pH is usually about 5-8, preferably, the pHis about 6-8.

As used herein, the term “effective amount” or “effective dose” refersto an amount that can produce function or activity for feeding theinsect and can be accepted by the insect. Preferably, the content ofdsRNA1 is about 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably,50-150 ng/μl; the content of dsRNA2 is about 1-500 ng/μl, preferably,5-300 ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA3 isabout 1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150ng/μl; the content of dsRNA4 is about 1-500 ng/μl, preferably, 5-300ng/μl, more preferably, 50-150 ng/μl; the content of dsRNA5 is about1-500 ng/μl, preferably, 5-300 ng/μl, more preferably, 50-150 ng/μl; thecontent of dsRNA6 is about 1-500 ng/μl, preferably, 5-300 ng/μl, morepreferably, 50-150 ng/μl. The selection of the preferred effectiveamount can be determined by a person of ordinary skill in the artaccording to various factors (for example, through a feeding experimentor a spray experiment).

As used herein, “insect feeding acceptable” ingredients are suitable forthe insects without excessive adverse side effects (such as toxicity,irritation, and allergic reactions), that is, substances with areasonable benefit/risk ratio.

As used herein, the term “carrier” includes various excipients anddiluents. Such carriers include (but are not limited to): water, saline,buffer, glucose, glycerol, ethanol, and combinations thereof.

The composition of the present invention can be directly sprayed, fed,or made into an injection form, for example, prepared by conventionalmethods with water, physiological saline or an aqueous solutioncontaining glucose and other adjuvants. The composition is preferablymanufactured under sterile or RNase-free conditions.

The main advantages of the present invention include:

1) The dsRNA designed for specific target genes of the present inventioncan effectively kill aphids, improve the control effect of aphids (≥80%)and the dropping rate of insect population (≥70%);

2) The obtained dsRNA can be directly used to kill aphids and isconvenient to use;

3) Low production cost, good stability, suitable for mass production;

4) Good environmental compatibility, green and pollution-free, and safefor humans and animals.

The invention will be further illustrated with reference to thefollowing specific examples. It is to be understood that these examplesare only intended to illustrate the invention, but not to limit thescope of the invention. For the experimental methods in the followingexamples without particular conditions, they are performed under routineconditions (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989) or as instructedby the manufacturer. Unless otherwise specified, the materials andreagents used in the examples are all commercially available products.

General Methods and Materials

1. Aphid Breeding and Biological Testing

The green peach aphid (Myzus persicae) was cultivated and tested onradish seedlings cultivated in indoor greenhouses or plasticgreenhouses, and the soybean aphid (Aphid glycine) was cultivated andtested on soybean seedlings cultivated in indoor greenhouses or plasticgreenhouses. The temperature of the incubation room was 25±1° C., therelative humidity was 40-60%, and the photoperiod was 12 h: 12 h.

Before the test, a certain number of aphids were inoculated on thetarget plant and counted. After spraying a certain concentration ofdsRNA, the counting was performed on day 1, 3, and 5 respectively. Thetest for each gene was repeated 10 times. According to the countingresults, the control effect of the target gene was determined.

2. Statistical Methods of Control Effect

In this study, two statistical methods were used to evaluate the effectof target dsRNA on aphids.

The first method, the control effect, is calculated as follows:

Control effect (%)=(1−CK0×PT1/CK1×PT0)×100

wherein PT0: the number of insects before drug administration in thetreatment area; PT1: the number of insects after drug administration inthe treatment area;

-   -   CK0: the number of insects before drug administration in the        control area; CK1: the number of insects after drug        administration in the control area.

The second method, the dropping rate of insect population, is calculatedas follows:

the dropping rate of insect population (%)=[(number of insects beforedrug administration−number of insects after drug administration)/numberof insects before drug administration]×100

3. RNA Extraction and Quality Test

The total RNA was extracted using TRIzol® Reagent (Invitrogen), and theoperation was performed according to the instructions: 1) adding 50-100mg of Ostrinia nubilalis sample that was well ground into 1 mL ofTRIzol, mixed well, and placed at room temperature for 5 minutes. 2)adding 200 μL of chloroform, shaked and mixed, and placed at roomtemperature for 3 minutes. 3) centrifuged at 12,000 rpm (4° C.) for 15minutes, transferring the upper aqueous phase to another new centrifugetube, adding 500 μL of pre-cooled isopropanol, shaked and mixed, andplaced at room temperature for 10 minutes. 4) centrifuged at 12 000 rpm(4° C.) for 15 minutes, and carefully aspirating the supernatant. 5)washed with 500 μL of pre-cooled 75% ethanol and mixed gently with avortex for 10 sec. 6) centrifuged at 12 000 rpm (4° C.) for 2 minutes,carefully aspirating the supernatant and drying it at room temperaturefor 5 minutes, adding an appropriate amount of DEPC sterilized water todissolve it, and obtaining a total RNA sample. Detecting the absorbanceunder a spectrophotometer, detecting the total RNA quality by 1% agargel electrophoresis, and storing it at −80° C., ready for use.

4. dsRNA Synthesis

Using the kit MEGAscript® RNAi Kit (Ambion) to synthesize dsRNA, andperforming experimental operations according to the instructions. A T7promoter sequence was added to the 5′end of the primer of theamplification template to facilitate subsequent dsRNA synthesis. UsingpPigbac A3 EGFP as a template for the synthesis of control group dsEGFP,dsEGFP was used as a negative control to participate in the treatment ofthe experimental group in subsequent experiments. See Appendix S3 forthe primers used to synthesize dsRNAs. During the synthesis process,template DNA and single-stranded RNA were removed with DNase and RNase,respectively.

5. Detection of Gene Expression (q-RT-PCR)

Using TRIzol® reagent (Invitrogen) for total RNA extraction, and thesteps were strictly in accordance with the operation manual. Taking 1 μgof total RNA and using the kit ReverTra Ace® qPCR RT Master Mix withgDNA Remover (TOYOBO) to synthesize the first strand of cDNA. The kitused for the RT-qPCR reaction was SYBR® Premix Ex Taq™ II (Takara), andthe primers were detailed in Appendix S3. For each gene sample, thedetection was repeated 3 times, the expression level analysis selectedthe expression level of 18S rRNA for normalization. Data analysis wasreferred to 2^(−ΔΔCT) Method (Livak & Schmittgen, 2001). Thecorresponding value was obtained by calculating the mean value andstandard error. In order to eliminate individual differences, thesamples of each experimental group were a sample pool formed by 2surviving larvae after treatment, and each experimental group wassubjected to three biological replicates.

Example 1 Target Gene Sequence and dsRNA Synthesis

In order to screen effective target genes of aphids based on RNAinterference technology, transcriptome sequencing was performed on thegreen peach aphid (Myzus persicae) and the soybean aphid (Aphid glycine)(the sampling and sequencing analysis methods of the two aphids weresame). After extracting total RNA from aphids at different developmentalstages, the same amount of RNA was taken and mixed to form the total RNAfor the entire developmental stage of aphids and sent to Shenzhen BGITechnology Services Co. LTD for transcriptome sequencing using theIllumina Hiseq2000 platform. After removing the adapters from thesequencing results, using the denove program to assemble, and thenperforming functional annotations on Unigene. In this study, target genefragments were selected from these functionally annotated Unigenes foramplification and dsRNA was synthesized. Through a large number ofscreenings, the primers of the present invention for the amplificationand synthesis of 6 target genes, the exogenous control gene GFP and theendogenous control gene DS50 from soybean aphid were shown in Table 1.The DNA sequences of the 6 gene fragments were shown in Table 2. Whereinds7, ds9, and ds15 were against aphids, especially the green peachaphid, and ds25, ds27, and ds45 were against aphids, especially thesoybean aphid.

TABLE 1 Amplification and synthesis of the primersequence of the target gene dsRNA. SEQ SEQ ID ID Name Primer F NO.:Primer R NO.: dsGFP TAATAC GACTCA CTATAG GGAGA 7TAATAC GACTCA CTATAG GGAGA 8 GACGAC GGCAAC TACA ACTCCA GCAGGA CCAT ds7TAATAC GACTCA CTATAG GGAGA 9 TAATAC GACTCA CTATAG GGAGA 10TCGCCA TCTACC CAGCCC CT CGGGTA CCACGG TTGGGG GT ds9TAATAC GACTCA CTATAG GGAGA 11 TAATAC GACTCA CTATAG GGAGA 12GCCGGT GGTATC TCCGCT GC TGGGGT CTGGCA ACATTC CCT ds15TAATAC GACTCA CTATAG GGAGA 13 TAATAC GACTCA CTATAG GGAGA 14TGGTGA ACCATT GGGCCG TGG AGGCGC ACGCTT GGGAAT GA ds25TAATAC GACTCA CTATAG GGAGA 15 TAATAC GACTCA CTATAG GGAGA 16CCACCT GGCATG GACGTC GG ACGTCC TCGGGC ATTGTA GCA ds27TAATAC GACTCA CTATAG GGAGA 17 TAATAC GACTCA CTATAG GGAGA 18GCGCAA GGTTAT GCGAGC GG CGGTTA GGGCTT CGGGGT CG ds45TAATAC GACTCA CTATAG GGAGA 19 TAATAC GACTCA CTATAG GGAGA 20GCAGAA GCCCGT GGCCCA AA TAGGCG CACCCA TCCCAC GA ds50TAATAC GACTCA CTATAG GGAGA 21 TAATAC GACTCA CTATAG GGAGA 22CGTGTC TGAGGC GGTTGC CA TGATCT TGGCCC GGAGAG CCGG

TABLE 2 Sequence fragments of 6 target genes. SEQ ID Name Sequence NO.:ds7 >CL1054.Contig1_TY tubulin alpha 1 chain-likeATGCGTGAATGTATCTCTGTACACGTTGGCCAA GCTGGTGTTCAAATCGGTAATGCCTGCTGGGAATTGTACTGTTTGGAACATGGAATTGCTCCAGAT GGTCAAATGCCATCTGACAAGACCATTGGAGGTGGAGACGACAGCTTCAACACCTTCTTCAGCGA AACTGGCTCAGGCAAACATGTGCCAAGAGCTGTGTTCGTTGATCTCGAACCAACTGTTGTTGATG AGGTAAGAACTGGAACATACCGCCAGTTGTTCCACCCTGAACAATTGATCACTGGTAAGGAAGAT GCCGCCAACAACTACGCACGTGGACACTACACTATCGGAAAAGAGATTGTTGATGTTGTTTTGGA CCGAATCAGGAAATTGGCTGATCAGTGCACTGGTCTTCAAGGTTTCCTGATCTTCCACTCTTTCGG AGGTGGTACTGGATCTGGTTTCACATCTTTGTTGATGGAAAGACTCAGCGTTGACTACGGAAAGA AGAGTAAATTAGAATTCGCCATCTACCCAGCCCCTCAAGTATCCACAGCTGTAGTTGAGCCATACA ACTCCATCTTGACCACACATACAACTCTTGAACACAGTGACTGTGCATTCATGGTCGATAATGAAG CCATCTATGACATCTGCCGTCGTAATCTCGATATTGAACGTCCAACTTACACTAACTTGAATCGTC TTATTGGCCAGATTGTTTCTTCAATCACAGCTTCTCTCCGTTTCGATGGTGCCCTCAATGTTGACTTG ACTGAATTCCAGACCAATTTGGTCCCATACCCCCGTATTCATTTCCCATTGGTCACCTATGCACCA GTCATCTCCGCTGAAAAGGCTTACCATGAACAATTGTCCGTATCAGAAATCACTAACGCTTGTTTT GAACCAGCCAACCAAATGGTGAAATGTGATCCACGTCATGGCAAATACATGGCTTGTTGCATGTT GTACCGTGGTGATGTTGTACCCAAAGACGTCAACGCTGCCATTGCTTCCATCAAGACCAAGAGAAC AATTCAGTTTGTTGACTGGTGTCCAACTGGTTTCAAAGTTGGTATCAACTACCAACCCCCAACCGT GGTACCCGGTGGTGACTTGGCTAAGGTACAACGTGCCGTCTGCATGTTGTCCAACACTACAGCTA TTGCTGAAGCTTGGGCTAGGTTGGACCACAAGTTCGACTTGATGTACGCCAAACGTGCTTTCGTCC ATTGGTATGTTGGAGAAGGTATGGAAGAAGGAGAATTCTCTGAAGCTCGTGAGGATTTGGCTGCT CTAGAGAAAGATTACGAAGAGGTTGGCATGGACTCCGTCGAAGGCGAAGGCGAAGGTGGTGAAG AATAC ds9 >CL3025.Contig1_TY ADP/ATP 2translocase 3-like ATGGCCGAAACCAAAGCGCCGAAGGACCCGTATGGTTTCTTGAAGGACTTCATGGCCGGTGGTAT CTCCGCTGCCGTGTCGAAGACCGCCGTGGCTCCGATCGAGCGCGTCAAGCTTATCCTGCAAGTGCA GGCCGCTTCCACGCAGATCGCCGCCGACCAACAGTACAAAGGAATTATGGACTGTTTGGTGAGA ATCCCAAAAGAACAAGGATTTGCCAGTTTCTGGAGAGGTAACTTTGCCAATGTCATCAGGTACTTC CCAACACAAGCATTGAACTTTGCTTTCAAGGATGTCTACAAACAGGTGTTTATGGACGGTGTGGAT AAAAAGACTCAATTCTGGCGGTATTTTGCTGGTAACTTGGCATCTGGTGGTGCTGCTGGAGCAACA TCTTTGTGCTTTGTATACCCCCTCGATTACGCACGTACACGATTAGGAGCTGATGTCGGTAAAGGA CCAGCTGAAAGGCAGTTCAAAGGTCTTGGTGATTGTTTAGCCAAAACCGTCAAGTCTGATGGTCCC ATTGGTTTGTACCGTGGTTTCATTGTATCAGTACAGGGTATCATCATCTACCGTGCTGCATACTTTG GATTTTTCGACACAGCTAAGGGAATGTTGCCAGACCCCAAGAATACTCCATTCTTAGTTTCATGGG GTATCGCCCAATTTGTAACAACATTCGCTGGTATTATGTCCTATCCATTTGACACAGTCAGACGTC GTATGATGATGCAATCTGGCCGTGCTGCTGACCAACGCATGTACAAGAGCACATTGGACTGCTGG GGTAAACTTTACAAGAATGAAGGTACATCTGCTTTCTTCAAGGGTGCATTCTCCAACGTACTCAGA GGTACTGGTGGTGCCTTGGTGTTGGTCTTCTACGACGAACTCAAAAACCTCATG ds15 >CL597.Contig1_TY heat shock 3protein 83-like ATGCCTGAAGACGTTACCATGACTGCATCTGATGATGTTGAGACCTTCGCTTTCCAAGCTGAGATC GCTCAGCTTATGTCCCTCATCATCAACACCTTCTACTCGAACAAAGAAATCTTTTTGCGAGAATTGG TATCCAATTCTTCTGATGCATTGGACAAAATTCGTTATGAGTCATTGACTGATCCATCCAAATTGG AATCTGGCAAAGATTTACACATTAAAATCATCCCCAATGCGGAAGAAAAAACTCTGACCATTATT GACACTGGTATCGGTATGACCAAAGCTGATCTAGTCAACAACTTGGGAACCATTGCTAAATCTGGT ACTAAGGCTTTCATGGAAGCTTTACAAGCTGGAGCTGATATTTCCATGATTGGTCAATTTGGTGTG GGTTTCTATTCCGCCTATCTGGTAGCTGACAAAGTCACTGTTGTTTCCAAACACAACGACGATGAA CAATATTTGTGGGAATCTGCTGCCGGAGGTTCATTCACCATCCGTACTGATCCTGGTGAACCATTG GGCCGTGGTACCAAAATTGTCCTTCAAATCAAAGAAGATCAAGCTGAGTTCCTCCAACAAGAAAA AATTACCAGCATCATCAAGAAGCACTCTCAATTCATTGGCTACCCAATCAAATTAATCGTTGAGAA TGAACGTACCAAAGAAGTCAGCGATGATGAAGCTGAAGAAGAAAAGAAAGATGAAGTTGAAGGT GAAACTGAAGAAGACAAAAAACCCAAAATTGAGGATGTTGGTGAGGATGAAGACGAAGACAAAA AAGATGAAGACAAAGACAAAAAGAAGAAGAAGACTATTAAAGAAAAGTACTTGGATGAAGAGG TCTTGAACAAGACAAAACCAATCTGGACACGCAACCCTGATGATATCAGCCAAGATGAATATGGT GAATTCTACAAATCCTTAACCAATGACTGGGAAGATCATTTAGCCGTCAAACATTTCTCTGTGGAA GGACAACTTGAATTCAGAGCATTGTTATTCATTCCCAAGCGTGCGCCTTATGACATGTTTGAGAAC AAGAAGAAGAAGAACAACATTAAATTATATGTCCGTCGTGTCTTCATCATGGACAACTGCGAAGA CCTCATGCCAGAATACTTGAACTTCATCAAGGGTGTTGTTGACAGTGAGGATTTGCCGTTGAACAT CTCCCGTGAAATGCTCCAACAAAACAAGATCTTGAAAGTTATCAGGAAGAATTTGGTTAAGAAAT GTTTGGAATTGTTCGAGGAATTGGCTGAAGACAAGGACAACTACAAGAAATTGTACGAACAGTTC AGCAAGAACTTGAAACTTGGAATCCACGAAGATAGCCAAAACAGAAAGAAACTCTCAGACTTGT TGAGATTCCACTCCTCAGCCAGTGGTGACGAATCATGCTCCCTTAAGGAGTATGTTGCACGTATGA AGCCAAATCAAACCCACATTTACTACATCACAGGTGAAAGCCGTGAACAAGTATCCAACTCTTCAT TCGTTGAACGTGTCAAGAAACGTGGTTTTGAAGTTATTTACATGACTGAACCCATTGATGAATACG TTGTCCAACAAATGAAAGAATATGACGGCAAGAACTTGGTATCTGTCACTAAAGAAGGTTTGGAC TTGCCTGAAACCGATGAAGAAAAGAAGAAGCGCGAGGATGATCAATCCAGATTTGAAAAATTGTG CAAAGTTGTTAAGGACATTTTGGACAAGAAAGTTGAGAAGGTTGTCATCAGTAACAGACTTGTTG AGTCTCCCTGTTGCATTGTCACATCTCAGTATGGTTGGACTGCCAACATGGAACGTATCATGAAG GCACAAGCACTCAGAGATTCATCTACCATGGGTTATATGTCTGCCAAAAAACACTTGGAAATCAAC CCTGACCACCCGATCATTGAAACACTCAGACAAAAGGCTGAAGCTGATTGCAACGACAAGGCTGT CAGAGACTTGGTCATGCTTTTGTTCGAGACAAGTTTGTTGTCATCTGGTTTTGGACTTGAAGACCC ACAAGTTCACGCTTCTAGAATCCACAGAATGATCAAATTGGGTTTGGGCATTGATGAAGATTTGCC AGTAGTTGAAGAAAAATCTGCTGAAGTTGAAGCCTCCGAGCCTGTTGTTGAAGCTGATGCTGAAG ATTCTTCTCGCATGGAAGAAGTTGATds25 >CL5923.Contig1_Ag_all eukaryotic 4 initiation factor 4A-likeATGAATGCTAATGAGACGAAAAATGGACCTCC TAGTGAAACCAATGACTACTCGGGACCACCTGGCATGGACGTCGGTGGAACTATTGAGTCTGACT GGAAAGAAGTGGTGGATAACTTTGATGAGATGAATTTAAAAGAAGAATTGTTGCGTGGTATTTAT GGATATGGTTTTGAAAAGCCATCAGCTATTCAACAACGTGCTATTTTGCCGTGCATCAAGGGACAT GATGTCATTGCTCAGGCCCAATCTGGTACTGGCAAGACAGCTACTTTTTCCATTTCTATTCTCCAAC AAATTGATACAAGTTTGAATGAGTGCCAAGCACTTATTTTGGCACCAACACGTGAATTGGCTCAA CAGATTCAAAAGGTGGTCATTGCTTTGGGTGATTTCATGAAAGCTGATTGTCATGCTTGCATTGGC GGTACAAACGTTCGTGATGACATGCGTAAGCTGGATACTGGATCCCATGTAGTTGTTGGAACTCCT GGCCGTGTTTATGACATGATTGCTAGAAAATCCCTAAGAACTCAATTTATCAAGATATTTGTGTTG GACGAAGCTGATGAAATGTTGTCTCGAGGTTTCAAAGATCAAATTAAAGAGGTGTTCAAGTTCCTC GAAGAAGATATTCAGGTCATTCTGTTGTCTGCTACAATGCCCGAGGACGTTTTGGATGTGAGCACT CATTTCATGCGTAATCCAGTACGCATTCTTGTTCAAAAGGAAGAACTGACATTGGAAGGTATCAAA CAGTTTTACATCAATGTTACCAAAGAAGAATGGAAGTTTGACACTCTATGTGATTTGTACGACACT CTTAGTATCACCCAGGCTGTGATCTTCTGTAACACACGTCGTAAGGTAGAGTGGTTGACTGAAAA TATGCGTTTGAAAACATTTACTGTATCAGCTATGCATGGAGAAATGGACCAACGTCAACGTGAGC TAATTATGCGTCAATTCCGTTCTGGCTCTAGTCGTGTTCTAATTACCACTGATTTGTTGGCTCGAG GCATTGATGTACAACAAGTTTCTCTGGTCATCAATTACGATTTGCCGTCCAATCGTGAAAACTATA TTCACAGGATTGGACGTTCTGGCCGTTTCGGTCGTAAAGGAGTCGCCATTAATTTTATCACCGAAG ACGACAAAAGAGCTATGAAGGATATTGAATCATTTTACAACACTCACGTGCTCGAGATGCCACAG AATGTGGCCGATTTGCTGds27 >CL6080.Contig1_Ag_all troponin 5 T-like isoform 3ATGTCCGACGAAGAAGAAGTGTACACTGATTC CGAAGAAGAAACGCAACCGGAGCCTGAAAAAAGCAAAGATGGAGATGGAGATCCCGAATTCGTT AAGAGGCAAGAATTAAAATCTTCAGCCTTAGACGAACAGCTTAAAGAGTACATCCAAGAATGGC GCAAACAGCGGTCAAAGGAAGAAGACGACTTAAAGAAGTTGAAGGAAAAACAGGCCAAGCGCAA GGTTATGCGAGCGGAAGAAGAGAAGAGAATGGCCGAGAGAAAGAAGCAAGAAGAAGAACGCAG ACAGAGAGAAGTCGAGGAAAAGAAACAAAAGGACATCGAAGAAAAACGTAAACGTCTAGAAGA GGCCGAGAAAAAACGGCAAGCTATGATGGCTGCTCTTAAGGAACAAACCAATAAATCTAAAGGA CCAAATTTCACCATCAGCAAAAAAGAAGGTGCGTTGAGTATGACTTCTGCCCAACTTGAACGCAA TAAAACCAGAGAACAGATCGAAGAAGAAAAGAAAATATCGTTGAGCTTCAGAATCAAACCTTTGA ATATTGAAGGATTCTCTGTGCAAAAACTCCAATTCAAAGCTACCGAACTCTGGGACCAGATCATCA AGTTGGAAACAGAAAAATACGATTTGGAGGAAAGGCAAAAGAGACAAGATTACGACTTGAAAGA GTTGAAAGAACGTCAGAAGCAACAACTCCGCCACAAGGCTCTGAAGAAAGGTCTCGACCCCGAA GCCCTAACCGGCAAATACCCACCCAAGATCCAAGTCGCTTCCAAGTACGAGAGGCGAGTTGACA CGAGGTCTTATGATGACAAAAAGAAGCTGTTCGAAGGAGGTTATATGGAAACCACTAAAGAATC AATGGAAAAACAATGGACAGAAAAAAGTGACCAATTCGGTGGCCGCGCTAAAGGACGATTACCG AAATGGTTCGGCGAACGTCCGGGCAAGAAGAAGGATGACCCAGACACACCCGAAGAGGAAGAGC TCAAGAAAAACGAGGAAGACGAAGAACCGTTTGGCCTCGACGACGAAGAAGCTGAAGAAGAAGT TGAAGAGGAAGAAGAGGAGGAAGAAGAAGAGGAAGAGGAGGAGGAAGAGGAAGAAGAGGAAG AAGAAGAAGAGGAAGAGGAAGAAGAAGAAGA Ads45 >CL2125.Contig1_Ag_all Y-box 6 protein Ct-p40-likeATGGCGGAACAAGTCGGCGAGAGGAGGACGGA ACGGCCGCCGCAGAAGCCCGTGGCCCAAAAGCCGGTCATATCTGTGAAAGTCACCGGCGTTGTTA AATGGTTCAACGTCAAAAGCGGTTATGGTTTTATTAATCGTAATGATACAAAAGAAGATATATTTG TACATCAGTCTGCTATTATCAAGAACAACCCTAAGAAAATTGTACGCAGTGTCGGTGATGGAGAA ACTGTAGAATTTGACGTTGTTGAGGGCGAAAAAGGTCACGAAGCAGCAAATGTTACTGGTCCAG ATGGAGAAGCTGTTAAAGGATCACCTTATGCAGCTGAAAGAAGAAGAAATAACTATCGTCAGTG GTTTTATGGACGCCGTCCTAATACCCGTCCAAGAAATGGTGGTCAACCTCCAAGAGATGGTAGTC CAAGTGGTGACAAGGAAGAAACTGAAAATGAAGTAGGAGAACAACCAAGACGTTACCGCCAGCC ACGTCAACAGAATTGGTATAATAGCTATCGTGGAAATCGAAGAGGTCCACCACCAAATAGAGGAG AAGGTGGTGATTACAATGGTGGAGATAATTATGGATATGATAGTTCACCTCCTGGTAGAGGCAGA GGTCGTGGGATGGGTGCGCCTAGACGTTTCTTTAGACGTGGCAGTGGATTTAGAGGGAGCCGTGG AACAGGTGGTCCACCCAGAAGACCATATCAAGATGAAAATCAGGACAATGAATATAATCAAAGT GATGAAAATGGAGCAAATAGACCTCGTCCTCGCTATCGCCGCCGCAATAATCGTTCTAGAGCGAG AAGTGATGGTCCTCCAAGAGCCAATAGCCAAAGTGACAATGAATCTAAACAAAAAAACTTTGGA GGAGAAGCATTGGAACTGGATGAAAGTAGTCA TGCT

Example 2 the Control Effect of Target Gene dsRNA on Aphids

Inoculating a certain number of green peach aphid or soybean aphid onradish seedlings or soybean seedlings, first, recording the number ofaphids inoculated on each plant respectively, and dissolving thesynthesized dsRNA into 2% Tween-80, the dsRNA concentrations of the 6target genes were shown in Table 3. Then spraying 1 ml of dsRNA on theplants inoculated with aphids, and counting on the next day as thestatistical results of the first day after dsRNA treatment. Thencounting every other day for a total of 3 times and recording as theresults of the first day, the third day and the fifth day aftertreatment, using 2% Tween-80 and dsGFP as a control. The statisticalresults show that, compared with the control spraying only 2% Tween-80,the control effects of the 3 target genes of the green peach aphid andthe 3 target genes of the soybean aphid on the two kinds of aphids allhave exceeded 80% (FIG. 1, A, B).

TABLE 3 Spraying concentration of target gene dsRNA Gene name of greenpeach Concentration Gene name of Concentration aphid (ng/μl) soybeanaphid (ng/μl) dsGFP 295 dsGFP 265 ds7 233 ds25 282 ds9 241 ds27 257 ds15279 ds45 242

Example 3 Statistics of the Dropping Rate of Insect Population of Aphidsby Target Genes

Aphids are virginopara insects, born as first-instar newborn aphids. Theperiod from the first instar aphid to the time it can give birth isabout 5-7 days (affected by environmental temperature). There areobvious alternation of generations in aphids on a plant, that is,insects of different generations and sizes (different instars) exist atthe same time. Therefore, when the test plants are inoculated, therewill be aphids of various instars (such as 2th-4th, and there may beadults). In this way, after various test treatments, the aphids quicklybegin to reproduce and produce the next generation, resulting in thenumber of aphids on the tested plant being increased after countingbefore drug spraying. This is a great interference to the judgment ofthe control effect of aphids insecticides. Therefore, there is a morerigorous or relatively accurate calculation method for the controleffect of aphids, that is, the dropping rate of insect population (seethe general methods and materials section for the calculation formula).

The statistical results of the present invention show that afterspraying the dsRNA of the 3 green peach aphid target genes, the droppingrates of insect population of the green peach aphid population at 1 day,3 days and 5 days after treatment are shown in Table 4. The droppingrates of insect population have all reached more than 70% on the 5th dayafter spraying.

After spraying the dsRNA of the 3 soybean aphid target genes, thedropping rates of insect population of soybean aphid population at 1day, 3 days and 5 days after treatment are shown in Table 5. Thedropping rate of insect population on the 5th day after spraying, exceptfor ds45, the dropping rate of insect population of which is 67.61%, thedropping rates of insect population of the other two target genes areall above 70%.

TABLE 4 The dropping rate of insect population after spraying with dsRNAof target gene of green peach aphid Treatment 1 d 3 d 5 d CK −23.55 ±19.99  −43.45 ± 41.67  −111.98 ± 80.5    dsGFP 10.99 ± 33.56 −1.61 ±52.77 −58.05 ± 74.94  ds7 27.73 ± 16.59 61.37 ± 17.58 74.95 ± 9.11  ds929.18 ± 21.97 63.65 ± 11.32 70.35 ± 14.69 ds15 38.18 ± 22.79 62.66 ±19.69 71.17 ± 13.78

TABLE 5 the dropping rate of insect population after spraying with dsRNAof target gene of soybean aphid Treatment 1 d 3 d 5 d CK  5.03 ± 13.96−50.29 ± 29.93 −135.23 ± 62.03  dsGFP −0.75 ± 18.49 −28.57 ± 19.66−59.91 ± 29.55  ds25 24.02 ± 18.82 67.91 ± 22.1 73.85 ± 16.11 ds27 33.64± 23.56  69.28 ± 14.36 78.82 ± 10.62 ds45 24.59 ± 22.59  61.6 ± 21.4567.61 ± 25.64

Example 4 Comparison of the Control Effect of Green Peach Aphid Targetand Imidacloprid

Experimental Method:

1. Radish seedlings of 12-15 days, inoculated with 100 insects,stabilized for 1 day, sprayed with dsRNA the next day.

2. The concentration of dsRNA used for spraying was 300 ng/μl. Thesynthesized dsRNA was dissolved in water and sprayed 300 μl per plant.

3. The concentration of imidacloprid was 10,000 times solution (GermanyBayer Emerald 70% imidacloprid 3 g, water dispersible granules), sprayed300 μl per plant.

4. The experiment method: spray treatment, 4 replicates for eachtreatment.

The results are shown in Table 6, Table 7, and FIG. 3 and FIG. 4.

TABLE 6 Field test of green peach aphid: the dropping rate of insectpopulation 1 d 3 d 5 d Control −116.00 ± 15.06 Aa  −297.87 ± 132.86 Aa −518.78 ± 161.36 Aa Imidacloprid 37.33 ± 9.65 Bb  75.83 ± 15.25 Bb 84.35± 9.60 Bb dsGFP −67.29 ± 40.98 Aa  −241.98 ± 106.06 Aa  −369.79 ± 131.65Aa ds7 15.75 ± 27.55 Cb 51.54 ± 10.55 Cb 81.81 ± 9.47 Bb ds9 15.85 ±16.94 Cb 66.41 ± 11.20 Bb 72.64 ± 4.51 Cb ds15 21.15 ± 15.10 Bb 57.65 ±18.66 Bb 79.32 ± 4.92 Bb

TABLE 7 Field test of green peach aphid: Control effect 1 d 3 d 5 ddsGFP 22.85 ± 15.71 Aa  12.58 ± 12.90 Aa  22.88 ± 18.49 Aa ds7 61.30 ±11.62 Bb 86.26 ± 6.91 Bb 96.91 ± 1.49 Bb ds9 60.56 ± 10.51 Bb 91.25 ±2.22 Cb 95.18 ± 2.07 Bb ds15 63.67 ± 4.78 Bb  89.34 ± 3.21 Cb 96.51 ±1.12 Bb Imidacloprid 70.96 ± 4.25 Cc  93.78 ± 3.14 Cb 97.37 ± 1.88 Bb

The results show that the three genes for green peach aphid have shownobvious lethal effects on the third day, and the dropping rate of insectpopulation is over 70% on the fifth day. Compared with imidacloprid,there is no obvious difference in the dropping rate of insectpopulation. However, compared with the control dsGFP, the dropping rateof insect population shows a significant difference (Table 6); at thesame time, the statistical analysis of the control effect shows that thecontrol effect of these three target genes has reached more than 90%,and it can show better control effect on the third day (Table 7).Therefore, this result shows that these three target genes have a stronglethal effect on green peach aphid and can be used as target genes tocontrol green peach aphid for pest control.

Example 5 Detection of Target Gene Expression

In order to prove that the control effect of spraying dsRNA of thesetarget genes on aphids population is due to the inhibition of theexpression of target genes, aphids on day 1, 3, and 5 after treatmentwith 6 target genes dsRNA were collected, and quantitative PCR(q-RT-PCR) was used to detect whether the target gene was suppressed.The test results of the three target genes of green peach aphid areshown in FIG. 2A. Except for the ds9 gene, the target gene is induced tobe up-regulated after 1 day of treatment, all genes are significantlydown-regulated after 3 and 5 days of treatment, indicating that thedeath of aphids is closely related to the level of gene expression.

The detection results of the 3 soybean aphid target genes are shown inFIG. 2B. Except for the expression of the target gene after 1 day ofds25 gene treatment is not significantly different from the control, allgenes are significantly down-regulated after 3 and 5 days of treatment,indicating that the death of aphids is closely related to the level ofgene expression.

Example 6

Preparation of the Composition

This example provides a composition for efficiently killing aphids. Thecomposition is an aqueous solution and includes components:

-   -   1. The dsRNA for the DS7 gene fragment as shown in SEQ ID NO. 1        or 24 the concentration is 100 ng/μl;    -   2. The dsRNA for the DS9 gene fragment as shown in SEQ ID NO. 2        or 25 the concentration is 100 ng/μl.    -   3. The dsRNA for the DS15 gene fragment as shown in SEQ ID NO. 3        or 26 the concentration is 100 ng/μl.    -   4. The the dsRNA for the DS25 gene fragment as shown in SEQ ID        NO. 4 or 27 the concentration is 100 ng/μl.    -   5. The dsRNA for the DS27 gene fragment as shown in SEQ ID NO. 5        or 28 the concentration is 100 ng/μl.    -   6. The dsRNA for the DS45 gene fragment as shown in SEQ ID NO. 6        or 29 the concentration is 100 ng/μl.

Comparative Example 1

The method is the same as that of Examples 1 and 2, the difference isthat the target gene is DS50 and the primers used are:

primer F: (SEQ ID NO.: 30) TAATACGACTCACTATAGGGAGACGTGTCTGAGGCGGTTGCCAprimer R: (SEQ ID NO.: 31) TAATACGACTCACTATAGGGAGATGATCTTGGCCCGGAGAGCCGG

The length of the amplified product is 578 bp.

The DS50 gene is a fatty acid synthase-like gene. The sequence is shownin SEQ ID NO. 23, which encodes the FASN gene. Fatty acid synthase is amulti-enzyme protein that catalyzes fatty acid synthesis. It is not asingle enzyme, but an entire enzyme system composed of two identical 272kDa multifunctional polypeptides, in which the substrate is submittedfrom one functional domain to the next, and its main function is tocatalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA inthe presence of NADPH.

The results show that the dsRNA designed for the DS50 gene by the methodof the present invention has a very poor control effect on aphids, witha maximum of only about 23%.

The sequence of the DS50 gene fragment is shown in SEQ ID NO: 23:

(SEQ ID NO.: 23) TTGGAATTGATTCAACATCTAGCTCAAAGAGGAGCCCGCAAATTTGTTTTAGTGTCGAAATTGAACAACAAACCTCAGTCAGGTTACAAGACGTTGACCTTAAGACGGTTGAAGAACAAGAACGTTACCGTAGTCCTATCGTTTGCTGACCCATCAACAGTGAGAGGCGCTGAAGACGTACTGAGAGAAGCTGTAGCCCTCGGAACAGTCTGTGGTATTTACCACATAACCACCGCTCCGGAAACCAAACACTTGCAATCCCTGAGCGAAAAGGATTTCGCAGAGACGAAAAAAGTCGTGTCTGAGGCGGTTGCCAATTTGGACACACTGAGCAGGAGATTGATTCCTCAACTTGAATCGTTTGTTGTCCTTGCTCCGGCCGTCGCATCAAGAGGAGCTAAAGCCAAGTCCAACTACGTTTTCGCAAACGCAGATGTTATCAGAGTCGCTGAAGTCCGTAAAGTTTCGGGCTATCCAACAGTAGTCATAGAATACGGCGCAATCGAAGGTATTTCGAATGCGTTCAACAGTCCAAACTTCAAACCAGCGTCGATCGTTTCAGCGTTGAATGTTCTGGATGAAATTACCAAACAACCACAAAACCCAACAGTCGTGTCCTTCTCAAAATTCAACGGTCCAATTTATGAAGAAACGGATGCCGCCACTCCATTGTTGAAGACAATTGCCAAGATTTTCGGTTACAAGACACTGTCCCAAATTGAACAGACCTTTAATCTCGCTCAACTCGGCCTGGACACGTTCCTCGCACCACGCGTTCAAGAAGCCATCAGACAACAAGCCAACGCAGTCATCGAGGTAGAAGAACTAAGAACACTGACGTTCCCGGCTCTCCGGGCCAAGATCATC GAATTACTCGCC

All literatures mentioned in the present application are incorporated byreference herein, as though individually incorporated by reference.Additionally, it should be understood that after reading the aboveteaching, many variations and modifications may be made by the skilledin the art, and these equivalents also fall within the scope as definedby the appended claims.

1. A dsRNA construct, wherein the dsRNA construct is double-stranded,and its positive or negative strand contains a structure as shown inFormula I:Seq_(forward)-X-Seq_(reverse)  Formula I wherein Seq_(forward) is anucleotide sequence of insect nymph and/or adult stageregulation-related gene or fragment; Seq_(reverse) is a nucleotidesequence that is basically complementary to Seq_(forward); X is anintervening sequence between the Seq_(forward) and the Seq_(reverse),and the intervening sequence is not complementary to the Seq_(forward)and the Seq_(reverse), wherein the insect nymph and/or adult stageregulation-related gene is selected from the group consisting of DS7gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45 gene and acombination thereof.
 2. A dsRNA as shown in Formula II,

wherein Seq′_(forward) is a RNA sequence or sequence fragmentcorresponding to a nucleotide sequence of an insect nymph and/or adultstage regulation-related gene or fragment; Seq′_(reverse) is a sequencethat is basically complementary to the Seq′_(forward); X′ is none; or isan intervening sequence located between Seq′_(forward) andSeq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse); wherein, the insect nymph and/oradult stage regulation-related gene is selected from the groupconsisting of: DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45gene, and a combination thereof; ∥ represents the hydrogen bond formedbetween Seq forward and Seq_(reverse).
 3. The dsRNA of claim 1, theinsect is a phytophagous insect, preferably a homoptera insect, mostpreferably Aphis.
 4. An expression vector containing the dsRNA constructof claim
 1. 5. A host cell that contains an expression vector containingthe dsRNA construct of claim 1 or having the DNA sequence correspondingto the dsRNA construct integrated into the chromosome.
 6. A compositioncomprising the dsRNA construct of claim 1, and an acceptable carrier forinsect feeding.
 7. A method of: (1) improving the control effect ofaphids; (2) increasing the dropping rate of insect population; (3)decreasing the expression level of nymph and/or adult stageregulation-related gene; (4) reducing the initial number of insectpopulation; (5) reducing plant damage rate; and/or (6) reducing cropdamage degree and improving the quality of crop products, the methodcomprising administering the dsRNA construct of claim
 1. 8. A method forkilling insects, comprising the steps of: using an interference moleculethat interferes with the expression of an insect nymph and/or adultstage regulation-related gene, or feeding or spraying an insect with avector, cell, plant tissue or insect prevention and control reagentcontaining the interference molecule; preferably, the insect nymphand/or adult stage regulation-related gene is selected from the groupconsisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45gene, and a combination thereof.
 9. A method for preparing the dsRNA ofclaim 2, comprising the steps: (i) preparing a construct expressingdsRNA, and the construct is double-stranded, and its positive ornegative strand contains a structure as shown in Formula I:Seq_(forward)-X-Seq_(reverse)  Formula I wherein Seq_(forward) is anucleotide sequence of insect nymph and/or adult stageregulation-related gene or fragment; Seq_(reverse) is a nucleotidesequence that is basically complementary to Seq_(forward); X is anintervening sequence located between the Seq_(forward) and theSeq_(reverse), and the intervening sequence is not complementary to theSeq_(forward) and the Seq_(reverse), wherein the insect nymph and/oradult stage regulation-related gene is selected from the groupconsisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene, DS45gene and a combination thereof; (ii) transforming the construct asdescribed in step (i) into a host cell, thereby expressing and forming adsRNA as shown in Formula II in the host cell,

wherein Seq′_(forward) is a RNA sequence or sequence fragmentcorresponding to the Seq_(forward) sequence; Seq′_(reverse) is asequence that is basically complementary to the Seq′_(forward); X′ isnone; or is an intervening sequence located between Seq′_(forward) andSeq′_(reverse), and the intervening sequence is not complementary toSeq′_(forward) and Seq′_(reverse), ∥ represents the hydrogen bond formedbetween Seq_(forward) and Seq_(reverse).
 10. A method for preparing aninsect prevention and control reagent comprising the steps of: sprayingthe dsRNA construct of claim 1 on the surface of the plant, therebyproducing the insect prevention and control agent.
 11. A method forimproving a plant resistance to an insect, comprising: expressing arecombinant DNA construct in a plant, wherein the recombinant DNAconstruct comprises DNA encoding RNA, and the RNA has a sequence that issubstantially identical or substantially complementary to at least 21 ormore consecutive nucleotides of the target gene, wherein the target geneis an insect nymph and/or adult stage regulation-related gene, selectedfrom the group consisting of DS7 gene, DS9 gene, DS15 gene, DS25 gene,DS27 gene, DS45 gene, and a combination thereof.
 12. A method forpreparing a transgenic plant cell, comprising the steps: (i) introducingor transfecting a recombinant DNA construct into a plant cell so thatthe plant cell contains the construct, thereby producing the transgenicplant cell, wherein the recombinant DNA construct contains DNA encodingRNA, the RNA has a sequence that is substantially identical orsubstantially complementary to at least 21 or more consecutivenucleotides of the target gene, wherein the target gene is an insectnymph and/or adult stage regulation-related gene, selected from thegroup consisting of DS7 Gene, DS9 gene, DS15 gene, DS25 gene, DS27 gene,DS45 gene, and a combination thereof.
 13. A method for preparing atransgenic plant, comprising the steps: regenerating a transgenic plantcell prepared by the method of claim 12 into a plant body, therebyobtaining the transgenic plant.
 14. A composition comprising the dsRNAof claim 2, and an acceptable carrier for insect feeding.
 15. A methodof: (1) improving the control effect of aphids; (2) increasing thedropping rate of insect population; (3) decreasing the expression levelof nymph and/or adult stage regulation-related gene; (4) reducing theinitial number of insect population; (5) reducing plant damage rate;and/or (6) reducing crop damage degree and improving the quality of cropproducts, the method comprising administering the dsRNA construct ofclaim
 2. 16. A method of: (1) improving the control effect of aphids;(2) increasing the dropping rate of insect population; (3) decreasingthe expression level of nymph and/or adult stage regulation-relatedgene; (4) reducing the initial number of insect population; (5) reducingplant damage rate; and/or (6) reducing crop damage degree and improvingthe quality of crop products, the method comprising administering thehost cell of claim
 5. 17. A method of: (1) improving the control effectof aphids; (2) increasing the dropping rate of insect population; (3)decreasing the expression level of nymph and/or adult stageregulation-related gene; (4) reducing the initial number of insectpopulation; (5) reducing plant damage rate; and/or (6) reducing cropdamage degree and improving the quality of crop products, the methodcomprising administering the composition of claim
 6. 18. A method of:(1) improving the control effect of aphids; (2) increasing the droppingrate of insect population; (3) decreasing the expression level of nymphand/or adult stage regulation-related gene; (4) reducing the initialnumber of insect population; (5) reducing plant damage rate; and/or (6)reducing crop damage degree and improving the quality of crop products,the method comprising administering the composition of claim
 14. 19. Amethod for preparing an insect prevention and control reagent comprisingthe steps of: spraying the dsRNA of claim 2 on the surface of the plant,thereby producing the insect prevention and control agent.
 20. A methodfor preparing an insect prevention and control reagent comprising thesteps of: spraying the composition of claim 6 on the surface of theplant, thereby producing the insect prevention and control agent.